Compositions and methods for hydroxylating epothilones

ABSTRACT

Isolated nucleic acid sequences and polypeptides encoded thereby for epothilone B hydroxylase and mutants and variants thereof and a ferredoxin located downstream from the epothilone B hydroxylase gene are provided. Also provided are vectors and cells containing these vectors. In addition, methods for producing recombinant microorganisms, methods for using these recombinant microorganism to produce hydroxyalkyl-bearing epothilones and an epothilone analog produced by a mutant of epothilone B hydroxylase are provided.

BASIS FOR PRIORITY CLAIM

This application is a divisional application of U.S. application Ser. No. 10/321,188 filed Dec. 17, 2002 now U.S. Pat. No. 6,884,608, and claims the benefit of U.S. Provisional Application No. 60,344,271, filed December 26, 2001, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to isolated nucleic acids sequences and polypeptides encoded thereby for epothilone B hydroxylase and mutants and variants thereof, and a ferredoxin located downstream from the epothilone B hydroxylase gene. The present invention also relates to recombinant microorganisms expressing epothilone B hydroxylase or a mutant or variant thereof and/or ferredoxin which are capable of hydroxylating small organic molecule compounds, such as epothilones, having a terminal alkyl group to produce compounds having a terminal hydroxyalkyl group. Also provided are methods for recombinantly producing such microorganisms as well as methods for using these recombinant microorganisms in the synthesis of compounds having a terminal hydroxylalkyl group. The compositions and methods of the present invention are useful in preparation of epothilones having a variety of utilities in the pharmaceutical field. A novel epothilone analog produced using a mutant of epothilone B hydroxylase of the present invention is also described.

BACKGROUND OF THE INVENTION

Epothilones are macrolide compounds that find utility in the pharmaceutical field. For example, epothilones A and B having the structures:

have been found to exert microtubule-stabilizing effects similar to paclitaxel (TAXOL®) and hence cytotoxic activity against rapidly proliferating cells, such as, tumor cells or cells associated with other hyperproliferative cellular diseases, see Bollag et al., Cancer Res., Vol. 55, No. 11, 2325–2333 (1995).

Epothilones A and B are natural anticancer agents produced by Sorangium cellulosum that were first isolated and characterized by Hofle et al., DE 4138042; WO 93/10121; Angew. Chem. Int. Ed. Engl. Vol. 35, No13/14, 1567–1569 (1996); and J. Antibiot., Vol. 49, No. 6, 560–563 (1996). Subsequently, the total syntheses of epothilones A and B have been published by Balog et al., Angew. Chem. Int. Ed. Engl., Vol. 35, No. 23/24, 2801–2803, 1996; Meng et al., J. Am. Chem. Soc., Vol. 119, No. 42, 10073–10092 (1997); Nicolaou et al., J. Am. Chem. Soc., Vol. 119, No. 34, 7974–7991 (1997); Schinzer et al., Angew. Chem. Int. Ed. Eng., Vol. 36, No. 5, 523–524 (1997); and Yang et al., Angew. Chem. Int. Ed. Engl., Vol. 36, No. 1/2, 166–168, 1997. WO 98/25929 disclosed the methods for chemical synthesis of epothilone A, epothilone B, analogs of epothilone and libraries of epothilone analogs. The structure and production from Sorangium cellulosum DSM 6773 of epothilones C, D, E, and F was disclosed in WO 98/22461. FIG. 1 provides a diagram of the biotransformation as described in WO 00/39276 of epothilone B to epothilone F in Actinomycetes species strain SC15847 (ATCC PT-1043), subsequently identified as Amycolatopsis orientalis.

Cytochrome P450 enzymes are found in prokaryotes and eukaryotic cells and have in common a heme binding domain which can be distinguished by an absorbance peak at 450 nm when complexed with carbon monoxide. Cytochrome P450 enzymes perform a broad spectrum of oxidative reactions on primarily hydrophobic substrates including aromatic and benzylic rings, and alkanes. In prokaryotes they are found as detoxifying systems and as a first enzymatic step in metabolizing substrates such as toluene, benzene and camphor. Cytochrome P450 genes have also been found in biosynthetic pathways of secondary metabolites such as nikkomycin in Streptomyces tendae (Bruntner, C. et al, 1999, Mol. Gen. Genet. 262: 102–114), doxorubicin (Dickens, M. L, Strohl, W. R., 1996, J. Bacteriol, 178: 3389-3395) and in the epothilone biosynthetic cluster of Sorangium cellulosum (Julien, B. et al., 2000, Gene, 249: 153–160). With a few exceptions, the cytochrome P450 systems in prokaryotes are composed of three proteins; a ferredoxin NADH or NADPH dependent reductase, an iron-sulfur ferredoxin and the cytochrome P450 enzyme (Lewis, D. F., Hlavica, P., 2000, Biochim. Biophys. Acta., 1460: 353–374). Electrons are transferred from ferredoxin reductase to the ferredoxin and finally to the cytochrome P450 enzyme for the splitting of molecular oxygen.

SUMMARY OF THE INVENTION

An object of the present invention is to provide isolated nucleic acid sequences encoding epothilone B hydroxylase and variants or mutants thereof and isolated nucleic acid sequences encoding ferredoxin or variants or mutants thereof.

Another object of the present invention is to provide isolated polypeptides comprising amino acid sequences of epothilone B hydroxylase and variants or mutants thereof and isolated polypeptides comprising amino acid sequences of ferredoxin and variants or mutants thereof.

Another object of the present invention is to provide structure coordinates of the homology model of the epothilone B hydroxylase. The structure coordinates are listed in Appendix 1. This model of the present invention provides a means for designing modulators of a biological function of epothilone B hydroxylase as well as additional mutants of epothilone B hydroxylase with altered specificities.

Another object of the present invention is to provide vectors comprising nucleic acid sequences encoding epothilone B hydroxylase or a variant or mutant thereof and/or ferredoxin or a variant or mutant thereof. In a preferred embodiment, these vectors further comprise a nucleic acid sequence encoding ferredoxin.

Another object of the present invention is to provide host cells comprising a vector containing a nucleic acid sequence encoding epothilone B hydroxylase or a variant or mutant thereof and/or ferredoxin or a variant or mutant thereof.

Another object of the present invention is to provide a method for producing recombinant microorganisms that are capable of hydroxylating compounds, and in particular epothilones, having a terminal alkyl group to produce compounds having a terminal hydroxyalkyl group.

Another object of the present invention is to provide microorganisms produced recombinantly which are capable of hydroxylating compounds, and in particular epothilones, having a terminal alkyl group to produce compounds having a terminal hydroxyalkyl group.

Another object of the present invention is to provide methods for hydroxylating compounds in these recombinant microorganisms. In particular, the present invention provides a method for the preparation of hydroxyalkyl-bearing epothilones, which compounds find utility as antitumor agents and as starting materials in the preparation of other epothilone analogs.

Yet another object of the present invention is to provide a compound of Formula A:

referred to herein as 24-OH epothilone B or 24-OH EpoB, as well as compositions and methods for production of compositions comprising the compound of Formula A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic of the biotransformation as set forth in WO 00/39276, U.S. application Ser. No. 09/468,854, filed Dec. 21, 1999, of epothilone B to epothilone F by Amycolatopsis orientalis strain SC 15847 (PTA 1043).

FIG. 2 shows the nucleic acid sequence alignments of SEQ ID NO:5 through SEQ ID NO:22 used to design the PCR primers for cloning of the nucleic acid sequence encoding epothilone B hydroxylase.

FIG. 3 shows the sequence alignment between epothilone B hydroxylase (SEQ ID NO:2) and EryF (PDB code 1JIN chain A; SEQ ID NO:76). The asterisks indicate sequence identities, the colons (:) similar residues.

FIG. 4 provides a homology model of epothilone B hydroxylase based upon sequence alignment with EryF as shown in FIG. 3.

FIG. 5 shows an energy plot of the epothilone B hydroxylase model (indicated by dashed line) relative to EryF (PDB code 1JIN; indicated by solid line). An averaging window size of 51 residues was used, i.e., the energy at a given residue position is calculated as the average of the energies of the 51 residues in the sequence that lie with the given residue at the central positions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated nucleic acid sequences and polypeptides and methods for obtaining compounds with desired substituents at a terminal carbon position. In particular, the present invention provides compositions and methods for the preparation of hydroxyalkyl-bearing epothilones, which compounds find utility as antitumor agents and as starting materials in the preparation of other epothilone analogs.

The term “epothilone,” as used herein, denotes compounds containing an epothilone core and a side chain group as defined herein. The term “epothilone core,” as used herein, denotes a moiety containing the core structure (with the numbering of ring system positions used herein shown):

wherein the substituents are as follows:

Q is selected from the group consisting of

W is O or NR₆;

X is selected from the group consisting of 0, H and OR₇;

M is O, S, NR₈, CR₉R₁₀;

B₁ and B₂ are selected from the group consisting of OR₁₁, OCOR₁₂;

R₁–R₅ and R₁₂–R₁₇ are selected from the group consisting of H, alkyl, substituted alkyl, aryl, and heterocyclo, and wherein R₁ and R₂ are alkyl they can be joined to form a cycloalkyl;

R₆ is selected from the group consisting of H, alkyl, and substituted alkyl;

R₇ and R₁₁ are selected from the group consisting of H, alkyl, substituted alkyl, trialkylsilyl, alkyldiarylsilyl and dialkylarylsilyl;

R₈ is selected from the group consisting of H, alkyl, substituted alkyl, R₁₃C═O, R₁₄OC═O and R₁₅SO₂; and

R₉ and R₁₀ are selected from the group consisting of H, halogen, alkyl, substituted alkyl, aryl, heterocyclo, hydroxy, R₁₆C═O, and R₁₇OC═O.

The term “side chain group” refers to substituent G as defined above for Epothilone A or B or G₁ and G₂ as shown below.

G₁ is the following formula V HO—CH₂-(A₁)_(n)-(Q)_(m)-(A₂)_(o)  (V), and

G₂ is the following formula VI CH₃-(A₁)_(n)-(Q)_(m)-(A₂)_(o)  (VI),

where

A₁ and A₂ are independently selected from the group of optionally substituted C₁–C₃ alkyl and alkenyl;

Q is optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring; and

n, m, and o are integers independently selected from the group consisting of zero and 1, where at least one of m, n or o is 1.

The term “terminal carbon” or “terminal alkyl group” refers to the terminal carbon or terminal methyl group of the moiety either directly bonded to the epothilone core at position 15 or to the terminal carbon or terminal alkyl group of the side chain group bonded at position 15. It is understood that the term “alkyl group” includes alkyl and substituted alkyl as defined herein.

The term “alkyl” refers to optionally substituted, straight or branched chain saturated hydrocarbon groups of 1 to 20 carbon atoms, preferably 1 to 7 carbon atoms. The expression “lower alkyl” refers to optionally substituted alkyl groups of 1 to 4 carbon atoms.

The term “substituted alkyl” refers to an alkyl group substituted by, for example, one to four substituents, such as, halo, trifluoromethyl, trifluoromethoxy, hydroxy, alkoxy, cycloalkyloxy, heterocyclooxy, oxo, alkanoyl, aryloxy, alkanoyloxy, amino, alkylamino, arylamino, aralkylamino, cycloalkylamino, heterocycloamino, disubstituted amines in which the 2 amino substituents are selected from alkyl, aryl or aralkyl, alkanoylamino, aroylamino, aralkanoylamino, substituted alkanoylamino, substituted arylamino, substituted aralkanoylamino, thiol, alkylthio, arylthio, aralkylthio, cycloalkylthio, heterocyclothio, alkylthiono, arylthiono, aralkylthiono, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl, sulfonamido (e.g. SO₂NH₂), substituted sulfonamido, nitro, cyano, carboxy, carbamyl (e.g. CONH₂), substituted carbamyl (e.g. CONH alkyl, CONH aryl, CONH aralkyl or cases where there are two substituents on the nitrogen selected from alkyl, aryl or aralkyl), alkoxycarbonyl, aryl, substituted aryl, guanidino and heterocyclos, such as, indolyl, imidazolyl, furyl, thienyl, thiazolyl, pyrrolidyl, pyridyl, pyrimidyl and the like. Where noted above where the substituent is further substituted it will be with halogen, alkyl, alkoxy, aryl or aralkyl.

In accordance with one aspect of the present invention there are provided isolated polynucleotides that encode epothilone B hydroxylase, an enzyme capable of hydroxylating epothilones having a terminal alkyl group to produce epothilones having a terminal hydroxyalkyl group.

In accordance with another aspect of the present invention there are provided isolated polynucleotides that encode a ferredoxin, the gene for which is located downstream from the epothilone B hydroxylase gene. Ferredoxin is a protein of the cytochrome P450 system.

By “polynucleotides”, as used herein, it is meant to include any form of DNA or RNA such as cDNA or genomic DNA or mRNA, respectively, encoding these enzymes or an active fragment thereof which are obtained by cloning or produced synthetically by well known chemical techniques. DNA may be double- or single-stranded. Single-stranded DNA may comprise the coding or sense strand or the non-coding or antisense strand. Thus, the term polynucleotide also includes polynucleotides exhibiting at least 60% or more, preferably at least 80%, homology to sequences disclosed herein, and which hybridize under stringent conditions to the above-described polynucleotides. As used herein, the term “stringent conditions” means hybridization conditions of 60° C. at 2×SSC buffer. More preferred are isolated nucleic acid molecules capable of hybridizing to the nucleic acid sequence set forth in 1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 70, 72, or 74 or SEQ ID NO:3, or to the complementary sequence of the nucleic acid sequence set forth in SEQ ID NO:1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 70, 72, or 74 or SEQ ID NO:3, under hybridization conditions of 3×SSC at 65° C. for 16 hours, and which are capable of remaining hybridized to the nucleic acid sequence set forth in SEQ ID NO:1, 30, 32, 34, 36, 37, 38, 39, 40, 41, 42, 60, 62, 64, 66, 68, 70, 72 or 74 or SEQ ID NO:3, or to the complementary sequence of the nucleic acid sequence set forth in SEQ ID NO:1, 30, 32, 34, 36, 37, 38, 39, 40, 41 or 42, 60, 62, 64, 66, 68, 70, 72 or 74 or SEQ ID NO:3, under wash conditions of 0.5×SSC, 55° C. for 30 minutes.

In one embodiment, a polynucleotide of the present invention comprises the genomic DNA depicted in SEQ ID NO:1 or a homologous sequence or fragment thereof which encodes a polypeptide having similar activity to that of this epothilone B hydroxylase. Alternatively, a polynucleotide of the present invention may comprise the genomic DNA depicted in SEQ ID NO:3 or a homologous sequence or fragment thereof which encodes a polypeptide having similar activity to this ferredoxin. Due to the degeneracy of the genetic code, polynucleotides of the present invention may also comprise other nucleic acid sequences encoding this enzyme and derivatives, variants or active fragments thereof.

The present invention also relates to variants of these polynucleotides which may be naturally occurring, i.e., present in microorganisms such as Amycolatopsis orientalis and Amycolata autotrophica, or in soil or other sources from which nucleic acids can be isolated, or mutants prepared by well known mutagenesis techniques. Exemplary variants polynucleotides of the present invention are depicted in SEQ ID NO: 36–42.

By “mutants” as used herein it is meant to be inclusive of nucleic acid sequences with one or more point mutations, or deletions or additions of nucleic acids as compared to SEQ ID NO: 1 or 3, but which still encode a polypeptide or fragment with similar activity to the polypeptides encoded by SEQ ID NO: 1 or 3. In a preferred embodiment, mutations are made which alter the substrate specificity and/or yield of the enzyme. A preferred region of mutation with respect to the epothilone B hydroxylase gene is that region of the nucleic acid sequence coding for the approximately 113 amino acids residues comprising the active site of the enzyme. Also preferred are mutants encoding a polypeptide with at least one amino acid substitution at amino acid position GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU 231, SER294, PHE237, or ILE365 of SEQ ID NO:1. Exemplary polynucleotide mutants of the present invention are depicted in SEQ ID NO: 30, 32, 34, 60, 62, 64, 66, 68, 70, 72 and 74.

Cloning of the nucleic acid sequence of SEQ ID NO:1 encoding epothilone B hydroxylase was performed using PCR primers designed by aligning the nucleic acid sequences of six cytochrome P450 genes from bacteria. The following cytochrome P450 genes were aligned:

-   -   Sequence: Locus: STMSUACB; Accession number: M32238; Reference:         Omer, C. A., J. Bacteriol. 172: 3335–3345 (1990)     -   Sequence: Locus: STMSUBCB; Accession number: M32239; Reference:         Omer, C. A., J. Bacteriol. 172: 3335–3345 (1990)     -   Sequence: Locus: AB018074 (formerly STMORFA); Accession number:         AB018074; Reference: Ueda, K., J. Antibiot. 48: 638–646 (1995)     -   Sequence: Locus: SSU65940; Accession number: U65940; Reference:         Motamedi, H., J. Bacteriol. 178: 5243–5248 (1996)     -   Sequence: Locus: STMOLEP; Accession number: L37200; Reference:         Rodriguez, A. M., FEMS Microbiol. Lett. 127: 117–120 (1995)     -   Sequence: Locus: SERCP450A; Accession number: M83110; Reference:         Andersen, J. F. and Hutchinson, C. R., J. Bacteriol. 174:         725–735 (1992)

Alignments were performed using an implementation of the algorithm of Myers, E. W. and W. Miller. 1988. CABIOS 4:1, 11–17., the Align program from Scientific and Educational Software (Durham, N.C., USA). Three highly conserved regions were identified in the I-helix, containing the oxygen binding domain, in the K-helix, and spanning the B-bulge and L-helix containing the conserved heme binding domain. Primers were designed to the three conserved regions identified in the alignment. Primers P450-1⁺ (SEQ ID NO:23) and P450-1a⁺ (SEQ ID NO:24) were designed from the I helix, Primer P450-2⁺ (SEQ ID NO:25) was designed from the B-Bulge and L-helix region and Primer P450-3⁻ (SEQ ID NO:27) was designed as the reverse complement to the heme binding protein.

Genomic fragments were then amplified via polymerase chain reaction (PCR). After PCR amplification, the reaction products were separated by gel electrophoresis and fragments of the expected size were excised. The DNA was extracted from the agarose gel slices using the Qiaquick gel extraction procedure (Qiagen, Santa Clarita, Calif., USA). The fragments were then cloned into the PCRscript vector (Stratagene, La Jolla, Calif., USA) using the PCRscript Amp cloning kit (Stratagene). Colonies containing inserts were picked to 1–2 ml of LB broth with 100 μg/ml ampicillin, 30–37° C., 16–24 hours, 230–300 rpm. Plasmid isolation was performed using the Mo Bio miniplasmid prep kit (Mo Bio, Solano Beach, Calif., USA). This plasmid DNA was used as a PCR and sequencing template and for restriction digest analysis.

The cloned PCR products were sequenced using the Big-Dye sequencing kit from Applied Biosystems, (Foster City, Calif., USA) and were analyzed using the AB1310 sequencer (Applied Biosystems, Foster City, Calif., USA). The sequence of the inserts was used to perform a TblastX search, using the protocol of Altschul, S. F, et al., Mol. Biol. 215:403–410 (1990), of the non-redundant protein database. Unique sequences having a significant similarity to known cytochrome P450 proteins were retained. Using this approach, a total of nine different P450 sequences were identified from SC15847, seven from the genomic DNA template and two from the cDNA. Two P450 sequences were found in common between the DNA and cDNA templates. Of the fifty cDNA clones analyzed, two sequences were predominant, with twenty clones each. These two genes were then cloned from the genomic DNA.

The nucleic acid sequence of the genomic DNA was determined using the Big-Dye sequencing system (Applied Biosystems) and analyzed using an ABI310 sequencer. This sequence is depicted in SEQ ID NO:1. An open reading frame coding for a protein of 404 amino acids and a predicted molecular weight of 44.7 kDa was found within the cloned BglII fragment. The deduced amino acid sequence of this polypeptide is depicted in SEQ ID NO: 2. The amino acid sequence of this polypeptide was found to share 51% identity with the NikF protein of Streptomyces tendae (Bruntner, C. et al, 1999, Mol. Gen. Genet. 262: 102–114) and 48% identity with the Sca-2 protein of S. carbophilus (Watanabe, I. Et al, 1995, Gene 163: 81–85). Both of these enzymes belong to the cytochrome P450 family 105. The invariable cysteine found in the heme-binding domain of all cytochrome P450 enzymes is found at residue 356. This gene for epothilone B hydroxylase has been named ebh. The ATG start codon of a putative ferredoxin gene of 64 amino acids is found nine basepairs downstream from the stop codon of ebh. This enzyme was found to share 50% identity with ferredoxin genes of S. griseoulus (O'Keefe, D. P., et al, 1991, Biochemistry 30: 447–455) and S. noursei (Brautaset, T., et al, 2000, Chem. Biol. 7: 395–403). The nucleic acid sequence encoding this ferredoxin is depicted in SEQ ID NO:3 and the amino acid sequence for this ferredoxin polypeptide is depicted in SEQ ID NO:4.

The ebh gene sequence was also used to isolate variant cytochrome P450 genes from other microorganisms. Exemplary variant polynucleotides ebh43491, ebh14930, ebh53630, ebh53550, ebh39444, ebh43333 and ebh35165 of the present invention and the species from which they were isolated are depicted in Table 1 below. The nucleic acid sequences for these variants are depicted in SEQ ID NO:36-42, respectively.

TABLE 1 Variant polynucleotides ATCC ID Species ebh gene designation 43491 Amycolatopsis orientalis ebh43491 14930 Amycolatopsis orientalis ebh14930 53630 Amycolatopsis orientalis ebh53630 53550 Amycolatopsis orientalis ebh53550 39444 Amycolatopsis orientalis ebh39444 43333 Amycolatopsis orientalis ebh43333 35165 Amycolatopsis orientalis ebh35165

The amino acid sequences encoded by the exemplary variants ebh43491, ebh14930, ebh53630, ebh53550, ebh39444, ebh43333 and ebh35165 are depicted in SEQ ID NO:43–49, respectively. Table 2 provides a summary of the amino acid substitutions of these exemplary variants.

TABLE 2 Amino acid Substitutions Position ebh Substitution ebh variant 100 Gly Ser ebh14930, ebh43333, ebh53550, ebh43491 101 Lys Arg ebh14930 130 Ile Leu ebh14930 192 Ser Gln ebh14930 224 Ser Thr ebh14930, ebh43333, ebh53550, ebh43491 285 Ile Val ebh14930, ebh43333, ebh53550, ebh43491 69 Ser Asn ebh43333 256 Val Ala ebh43333, ebh53550, ebh43491 93 Ala Ser ebh53550 326 Asp Glu ebh53550, ebh43491 333 Thr Ala ebh53550, ebh43491 133 Leu Met ebh43491 398 His Arg ebh39444

Mutations were also introduced into the coding region of the ebh gene to identify mutants with improved yield, and/or rate of bioconversion and/or altered substrate specificity. Exemplary mutant nucleic acid sequences of the present invention are depicted in SEQ ID NO:30, 32, 34, 60, 62, 64, 66, 68, 70, 72 and 74.

The nucleic acid sequence of SEQ ID NO:30 encodes a mutant ebh25-1 which exhibits altered substrate specificity. Plasmid pANT849ebh25-1 containing this mutant gene was deposited and accepted by an International Depository Authority under the provisions of the Budapest Treaty. The deposit was made on Nov. 21, 2002 to the American Type Culture Collection at 10801 University Boulevard in Manassas, Va. 20110-2209. The ATCC Accession Number is PTA-4809. All restrictions upon public access to this plasmid will be irrevocably removed upon granting of this patent application. The Deposit will be maintained in a public depository for a period of thirty years after the date of deposit or five years after the last request for a sample or for the enforceable life of the patent, whichever is longer. The above-referenced plasmid was viable at the time of the deposit. The deposit will be replaced if viable samples cannot be dispensed by the depository.

This S. lividans transformant identified in the screening of mutation 25 (primers NPB29-mut25f (SEQ ID NO:58) and NPB29-mut25r (SEQ ID NO:59)) was found to produce a product with a different HPLC elution time than epothilone B or epothilone F. A sample of this unknown was analyzed by LC-MS and was found to have a molecular weight of 523 (M.W.), consistent with a single hydroxylation of epothilone B. Plasmid DNA was isolated from the S. lividans culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29) (see Example 17). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh25-1 mutant was found to have two mutations resulting in changes in the amino acid sequence of the protein, asparagine 195 is changed to serine and serine 294 is changed to proline. The position targeted for mutation at codon 238 was found to have a two nucleotide change, which did not result in a change of the amino acid sequence of the protein. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:30 is depicted in SEQ ID NO:31.

The nucleic acid sequence of SEQ ID NO:32 encodes a mutant ebh10-53, which exhibits improved bioconversion yield. This S. lividans transformant identified in the screening of mutation 10 (primers NPB29-mut10f (SEQ ID NO:54) and NPB29-mut10r (SEQ ID NO:55)) produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. lividans culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29)(see Example 16). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh10-53 mutant was found to have two mutations resulting in changes in the amino acid sequence of the protein, glutamic acid 231 is changed to arginine and phenylalanine 190 is changed to tyrosine. The position 231 was the target of the mutagenesis, the change at residue 190 is an inadvertent change that is an artifact of the mutagenesis procedure. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:32 is depicted in SEQ ID NO:33.

The nucleic acid sequence of SEQ ID NO:34 encodes a mutant ebh24-16, which also exhibits improved bioconversion yield. This S. lividans transformant, ebh24-16 identified in the screening of mutation 24 (primers NPB29-mut24f (SEQ ID NO:56) and NPB29-mut24r (SEQ ID NO:57) also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. lividans culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh24-16 mutant was found to have two mutations resulting in changes in the amino acid sequence of the protein, phenylalanine 237 is changed to alanine and isoleucine 92 is changed to valine. The position 237 was the target of the mutagenesis, the change at residue 92 is an inadvertent change that is an artifact of the mutagenesis procedure. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:34 is depicted in SEQ ID NO:35.

The nucleic acid sequence of SEQ ID NO:60 encodes a mutant ebh24-16d8, which also exhibits improved bioconversion yield. This S. rimosus transformant, ebh24-16d8 identified in the screening of mutation 59 (primer NPB29mut59 (SEQ ID NO:70)) also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh24-16d8 mutant was found to have one mutation resulting in a change in the amino acid sequence of the protein, arginine 67 is changed to glutamine. This change is an artifact of the mutagenesis procedure. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:60 is SEQ ID NO:61.

The nucleic acid sequence of SEQ ID NO:62 encodes a mutant ebh24-16c11, which also exhibits improved bioconversion yield. This S. rimosus transformant, ebh24-16c11 identified in the screening of mutation 59 (primer NPB29mut59 (SEQ ID NO:70)) also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh24-16c11 mutant was found to have two additional mutations resulting in changes in the amino acid sequence of the protein, alanine 93 is changed to glycine and isoleucine 365 is changed to threonine. The position 93 is the target of the mutagenesis, the change at 365 is an artifact of the mutagenesis procedure. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:62 is depicted in SEQ ID NO:63.

The nucleic acid sequence of SEQ ID NO:64 encodes a mutant ebh24-16-16, which also exhibits improved bioconversion yield. This S. rimosus transformant, ebh24-16-16 identified in the screening of random mutants of ebh24-16 also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh24-16-16 mutant was found to have one additional mutation resulting in changes in the amino acid sequence of the protein, valine 106 is changed to alanine. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:64 is depicted in SEQ ID NO:65.

The nucleic acid sequence of SEQ ID NO:66 encodes a mutant ebh24-16-74, which also exhibits improved bioconversion yield. This S. rimosus transformant, ebh24-16-74 identified in the screening of random mutants of ebh24-16 also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh24-16-74 mutant was found to have one additional mutation resulting in changes in the amino acid sequence of the protein, arginine 88 is changed to histidine. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:66 is SEQ ID NO:67.

The nucleic acid sequence of SEQ ID NO:68 encodes a mutant ebh24-M18, which also exhibits improved bioconversion yield. This S. rimosus transformant, ebhM-18 identified in the screening of random mutants of ebh also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebhM-18 mutant was found to have two mutations resulting in changes in the amino acid sequence of the protein, glutamic acid 31 is changed to lysine and methionine 176 is changed to valine. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:68 is depicted in SEQ ID NO:69.

The nucleic acid sequence of SEQ ID NO:72 encodes a mutant ebh24-16g8, which also exhibits improved bioconversion yield. This S. rimosus transformant, ebh24-16g8 identified in the screening of mutation 50 (primer NPB29mut50 (SEQ ID NO:71)) also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh24-16g8 mutant was found to have two additional mutations resulting in changes in the amino acid sequence of the protein, methionine 176 is changed to alanine and isoleucine 130 is changed to threonine. The position 176 is the target of the mutagenesis, the change at 130 is an artifact of the mutagenesis procedure. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:72 is depicted in SEQ ID NO:73.

The nucleic acid sequence of SEQ ID NO:74 encodes a mutant ebh24-16b9, which also exhibits improved bioconversion yield. This S. rimosus transformant, ebh24-16b9 identified in the screening of mutation 50 (primer NPB29mut50 (SEQ ID NO:71)) also produced a greater yield of epothilone F. Plasmid DNA was isolated from the S. rimosus culture and used as a template for PCR amplification using primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29). The expected fragment was obtained and sequenced using the Big-Dye sequencing system. The ebh24-16b9 mutant was found to have two additional mutations resulting in changes in the amino acid sequence of the protein, methionine 176 is changed to serine and alanine 140 is changed to threonine. The position 176 is the target of the mutagenesis, the change at 140 is an artifact of the mutagenesis procedure. The amino acid sequence of the mutant polypeptide encoded by SEQ ID NO:74 is depicted in SEQ ID NO:75.

A mixture composed of the plasmids pANT849ebh-24-16, pANT849ebh-10-53, pANT849ebh-24-16d8, pANT849ebh-24-16c11, pANT849ebh-24-16-16, pant849ebh-24-16-74, pANT849ebh-24-16b9, pANT849ebh-M18 and pANT849ebh-24-16g8 for these nine mutant genes was deposited and accepted by an International Depository Authority under the provisions of the Budapest Treaty. The deposit was made on Nov. 21, 2002 to the American Type Culture Collection at 10801 University Boulevard in Manassas, Va. 20110-2209. The ATCC Accession Number is PTA-4808. All restrictions upon public access to this mixture of plasmids will be irrevocably removed upon granting of this patent application. The deposit will be maintained in a public depository for a period of thirty years after the date of deposit or five years after the last request for a sample or for the enforceable life of the patent, whichever is longer. The above-referenced mixture of plasmids was viable at the time of the deposit. The deposit will be replaced if viable samples cannot be dispensed by the depository.

Thus, in accordance with another aspect of the present invention, there are provided isolated polypeptides of epothilone B hydroxylase and variants and mutants thereof and isolated polypeptides of ferredoxin or variants thereof. In one embodiment of the present invention, by “polypeptide” it is meant to include the amino acid sequence of SEQ ID NO: 2, and fragments or variants, which retain essentially the same biological activity and/or function as this epothilone B hydroxylase. In another embodiment of the present invention, by “polypeptide” it is meant to include the amino acid sequence of SEQ ID NO:4, and fragments and/or variants, which retain essentially the same biological activity and/or function as this ferredoxin.

By “variants” as used herein it is meant to include polypeptides with amino acid sequences with conservative amino acid substitutions as compared to SEQ ID NO: 2 or 4 which are demonstrated to exhibit similar biological activity and/or function to SEQ ID NO:2 or 4. By “conservative amino acid substitutions” it is meant to include replacement, one for another, of the aliphatic amino acids such as Ala, Val, Leu and Ile, the hydroxyl residues Ser and Thr, the acidic residues Asp and Glu, and the amide residues Asn and Gln. Exemplary variant amino acid sequences of the present invention are depicted in SEQ ID NO:43–49 and the amino acid substitutions of these exemplary variants are described in Table 2, supra.

By “mutants” as used herein it is meant to include polypeptides encoded by nucleic acid sequences with one or more point mutations, or deletions or additions of nucleic acids as compared to SEQ ID NO: 1 or 3, but which still have similar activity to the polypeptides encoded by SEQ ID NO: 1 or 3. In a preferred embodiment, mutations are made to the nucleic acid that alter the substrate specificity and/or yield from the polypeptide encoded thereby. A preferred region of mutation with respect to the epothilone B hydroxylase gene is that region of the nucleic acid sequence coding for the approximately 113 amino acid residues comprising the active site of the enzyme. Also preferred are mutants with at least one amino acid substitution at amino acid position GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU 231, SER294, PHE237, or ILE365 of SEQ ID NO:1 Exemplary mutants ebh25-1, ebh10-53, ebh24-16, ebh24-16d8, ebh24-16c11, ebh24-16-16, ebh24-16-74, ebh24-16g8, ebh24-16b9 and the nucleic acid sequences encoding such mutants of the present invention are depicted in SEQ ID NO:31, 33, 35, 61, 63, 65, 67, 69, 71, 73 and 75, and SEQ ID NO:30, 32, 34, 60, 62, 64, 66, 68, 70, 72 and 74, respectively.

A 3-dimensional model of epothilone B hydroxylase has also been constructed in accordance with general teachings of Greer et al. (Comparative modeling of homologous proteins. Methods In Enzymology 202239-52, 1991), Lesk et al. (Homology Modeling: Inferences from Tables of Aligned Sequences. Curr. Op. Struc. Biol. (2) 242–247, 1992), and Cardozo et al. (Homology modeling by the ICM method. Proteins 23, 403–14, 1995) on the basis of the known structure of a homologous protein EryF (PDB Code 1KIN chain A). Homology between these sequences is 34%. Alignment of the sequences of epothilone B hydroxylase (SEQ ID NO:2) and EryF (PDB Code 1KIN chain A; SEQ ID NO:76) is depicted in FIG. 3. A homology model of epothilone B hydroxylase based upon sequence alignment with EryF is depicted in FIG. 4.

An energy plot of the epothilone B hydroxylase model relative to EryF (PDB code 1JIN) was also prepared and is depicted in FIG. 5. An averaging window size of 51 residues was used at a given residue position to calculate the average of the energies of the 51 residues in the sequence that lie with the given residue at the central position. As shown in FIG. 5, all energies along the sequence lie below zero thus indicating that the modeled structure as set forth in FIG. 4 and Appendix 1 is reasonable.

The three-dimensional structure represented in the homology model of epothilone B hydroxylase of FIG. 4 is defined by a set of structure coordinates as set forth in Appendix 1. The term “structure coordinates” refers to Cartesian coordinates generated from the building of a homology model. As will be understood by those of skill in the art, however, a set of structure coordinates for a protein is a relative set of points that define a shape in three dimensions. Thus, it is possible that an entirely different set of coordinates could define a similar or identical shape. Moreover, slight variations in the individual coordinates, as emanate from generation of similar homology models using different alignment templates and/or using different methods in generating the homology model, will have minor effects on the overall shape. Variations in coordinates may also be generated because of mathematical manipulations of the structure coordinates. For example, the structure coordinates set forth in Appendix 1 could be manipulated by fractionalization of the structure coordinates; integer additions or subtractions to sets of the structure coordinates, inversion of the structure coordinates or any combination of the above.

Various computational analyses are therefore necessary to determine whether a molecule or a portion thereof is sufficiently similar to all or parts of epothilone B hydroxylase described above as to be considered the same. Such analyses may be carried out in current software applications, such as SYBYL version 6.7 or INSIGHTII (Molecular Simulations Inc., San Diego, Calif.) version 2000 and as described in the accompanying User's Guides.

For example, the superimposition tool in the program SYBYL allows comparisons to be made between different structures and different conformations of the same structure. The procedure used in SYBYL to compare structures is divided into four steps: 1) load the structures to be compared; 2) define the atom equivalencies in these structures; 3) perform a fitting operation; and 4) analyze the results. Each structure is identified by a name. One structure is identified as the target (i.e., the fixed structure); the second structure (i.e., moving structure) is identified as the source structure. Since atom equivalency within SYBYL is defined by user input, for the purpose of this aspect of the present invention equivalent atoms are defined as protein backbone atoms (N, Cα, C and O) for all conserved residues between the two structures being compared. Further, only rigid fitting operations are considered. When a rigid fitting method is used, the working structure is translated and rotated to obtain an optimum fit with the target structure. The fitting operation uses an algorithm that computes the optimum translation and rotation to be applied to the moving structure, such that the root mean square difference of the fit over the specified pairs of equivalent atoms is an absolute minimum. This number, given in angstroms, is reported by SYBYL.

For the purposes of the present invention, any homology model of epothilone B hydroxylase that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than about 4.0 Å when superimposed on the corresponding backbone atoms described by structure coordinates listed in Appendix 1 are considered identical. More preferably, the root mean square deviation is less than about 3.0 Å. More preferably the root mean square deviation is less than about 2.0 Å.

For the purpose of this invention, any homology model of epothilone B hydroxylase that has a root mean square deviation of conserved residue backbone atoms (N, Cα, C, O) of less than about 2.0 Å when superimposed on the corresponding backbone atoms described by structure coordinates listed in Appendix 1 are considered identical. More preferably, the root mean square deviation is less than about 1.0 Å.

In another embodiment of the present invention, structural models wherein backbone atoms have been substituted with other elements which when superimposed on the corresponding backbone atoms have low root mean square deviations are considered to be identical. For example, an homology model where the original backbone carbon, and/or nitrogen and/or oxygen atoms are replaced with other elements having a root mean square deviation of about 4.0 Å, more preferably about 3.0 Å, even more preferably less than about 2 Å, when superimposed on the corresponding backbone atoms described by structure coordinates listed in Appendix 1 is considered identical.

The term “root mean square deviation” means the square root of the arithmetic mean of the squares of the deviations from the mean. It is a way to express the deviation or variation from a trend or object. For purposes of this invention, the “root mean square deviation” defines the variation in the backbone of a protein from the relevant portion of the backbone of the epothilone B hydroxylase portion of the complex as defined by the structure coordinates described herein.

The present invention as embodied by the homology model enables the structure-based design of additional mutants of epothilone B hydroxylase. For example, using the homology model of the present invention, residues lying within 10 Å of the binding site of epothilone B hydroxylase have now been defined. These residues include LEU39, GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, LYS85, PRO86, PHE87, ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155, PHE169, GLN170, CYS172, SER173, SER174, ARG175, MET176, LEU177, SER178, ARG179, ARG186, PHE190, LEU193, VAL233, GLY234, LEU235, ALA236, PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283, THR287, ILE288, ALA289, GLU290, THR291, ALA292, THR293, SER294, ARG295, PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351, GLN352, CYS353, LEU354, GLY355, GLN356, LEU358, ALA359, GLU362, LYS389, ASP391, SER392, THR393, ILE394 and TYR395 as set forth in Appendix 1. Mutants with mutations at one or more of these positions are expected to exhibit altered biological function and/or specificity and thus comprise another embodiment of preferred mutants of the present invention. Another embodiment of preferred mutants are molecules that have a root mean square deviation from the backbone atoms of said epothilone B hydroxylase of not more than about 4.0 Å.

The structure coordinates of an epothilone B hydroxylase homology model or portions thereof are stored in a machine-readable storage medium. Such data may be used for a variety of purposes, such as drug discovery.

Accordingly, another aspect of the present invention relates to machine-readable data storage medium comprising a data storage material encoded with the structure coordinates set forth in Appendix 1.

The three-dimensional model structure of epothilone B hydroxylase can also be used to identify modulators of biological function and potential substrates of the enzyme. Various methods or combinations thereof can be used to identify such modulators.

For example, a test compound can be modeled that fits spatially into a binding site in epothilone B hydroxylase, according to Appendix 1. Structure coordinates of amino acids within 10 Å of the binding region of epothilone B hydroxylase defined by amino acids LEU39, GLN43, ALA45, MET57, LEU58, HIS62, PHE63, SER64, SER65, ASP66, ARG67, GLN68, SER69, LEU74, MET75, VAL76, ALA77, ARG78, GLN79, ILE80, ASP84, LYS85, PRO86, PHE87, ARG88, PRO89, SER90, LEU91, ILE92, ALA93, MET94, ASP95, HIS99, ARG103, PHE110, ILE155, PHE169, GLN170, CYS172, SER173, SER174, ARG175, MET176, LEU177, SER178, ARG179, ARG186, PHE190, LEU193, VAL233, GLY234, LEU235, ALA236, PHE237, LEU238, LEU239, LEU240, ILE241, ALA242, GLY243, HIS244, GLU245, THR246, THR247, ALA248, ASN249, MET250, LEU283, THR287, ILE288, ALA289, GLU290, THR291, ALA292, THR293, SER294, ARG295, PHE296, ALA297, THR298, GLU312, GLY313, VAL314, VAL315, GLY316, VAL344, ALA345, PHE346, GLY347, PHE348, VAL350, HIS351, GLN352, CYS353, LEU354, GLY355, GLN356, LEU358, ALA359, GLU362, LYS389, ASP391, SER392, THR393, ILE394 and TYR395, and the coordinated heme group, HEM1 can also be used to identify desirable structural and chemical features of such modulators. Identified structural or chemical features can then be employed to design or select compounds as potential epothilone B hydroxylase ligands. By structural and chemical features it is meant to include, but is not limited to, covalent bonding, van der Waals interactions, hydrogen bonding interactions, charge interaction, hydrophobic bonding interaction, and dipole interaction. Compounds identified as potential epothilone B hydroxylase ligands can then be synthesized and screened in an assay characterized by binding of a test compound to epothilone B hydroxylase, or in characterizing the ability of epothilone B hydroxylase to modulate a protease target in the presence of a small molecule. Examples of assays useful in screening of potential epothilone B hydroxylase ligands include, but are not limited to, screening in silico, in vitro assays and high throughput assays.

As will be understood by those of skill in the art upon this disclosure, other structure-based design methods can be used. Various computational structure-based design methods have been disclosed in the art. For example, a number of computer modeling systems are available in which the sequence of epothilone B hydroxylase and the epothilone B hydroxylase structure (i.e., atomic coordinates of epothilone B hydroxylase as provided in Appendix 1 and/or the atomic coordinates within 10 Å of the binding region as provided above) can be input. This computer system then generates the structural details of one or more these regions in which a potential epothilone B hydroxylase modulator binds so that complementary structural details of the potential modulators can be determined. Design in these modeling systems is generally based upon the compound being capable of physically and structurally associating with epothilone B hydroxylase. In addition, the compound must be able to assume a conformation that allows it to associate with epothilone B hydroxylase. Some modeling systems estimate the potential inhibitory or binding effect of a potential epothilone B hydroxylase substrate or modulator prior to actual synthesis and testing.

Methods for screening chemical entities or fragments for their ability to associate with a given protein target are also well known. Often these methods begin by visual inspection of the binding site on the computer screen. Selected fragments or chemical entities are then positioned in a binding region of epothilone B hydroxylase. Docking is accomplished using software such as INSIGHTII, QUANTA and SYBYL, following by energy minimization and molecular dynamics with standard molecular mechanic force fields such as, MMFF, CHARMM and AMBER. Examples of computer programs which assist in the selection of chemical fragment or chemical entities useful in the present invention include, but are not limited to, GRID (Goodford, 1985), AUTODOCK (Goodsell, 1990), and DOCK (Kuntz et al. 1982).

Upon selection of preferred chemical entities or fragments, their relationship to each other and epothilone B hydroxylase can be visualized and then assembled into a single potential modulator. Programs useful in assembling the individual chemical entities include, but are not limited to CAVEAT (Bartlett et al. 1989) and 3D Database systems (Martin 1992).

Alternatively, compounds may be designed de novo using either an empty active site or optionally including some portion of a known inhibitor. Methods of this type of design include, but are not limited to LUDI (Bohm 1992) and LeapFrog (Tripos Inc., St. Louis Mo.).

Programs such as DOCK (Kuntz et al. 1982) can be used with the atomic coordinates from the homology model to identify potential ligands from databases or virtual databases which potentially bind the in the active site binding region which may therefore be suitable candidates for synthesis and testing.

Also provided in the present invention are vectors comprising polynucleotides of the present invention and host cells which are genetically engineered with vectors of the present invention to produce epothilone B hydroxylase or active fragments and variants or mutants of this enzyme and/or ferredoxin or active fragments thereof. Generally, any vector suitable to maintain, propagate or express polynucleotides to produce these polypeptides in the host cell may be used for expression in this regard. In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single- or double-stranded phage vector, or a single- or double-stranded RNA or DNA viral vector. Vectors may be extra-chromosomal or designed for integration into the host chromosome. Such vectors include, but are not limited to, chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, and viruses such as baculoviruses, papova viruses, SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, cosmids and phagemids.

Useful expression vectors for prokaryotic hosts include, but are not limited to, bacterial plasmids, such as those from E. coli, Bacillus or Streptomyces, including pBluescript, pGEX-2T, pUC vectors, pET vectors, ColE1, pCR1, pBR322, pMB9, pCW, pBMS200, pBMS2020, PIJ101, PIJ702, pANT849, pOJ260, pOJ446, pSET152, pKC1139, pKC1218, pFD666 and their derivatives, wider host range plasmids, such as RP4, phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, λGT10 and λGT11, and other phages, e.g., M13 and filamentous single stranded phage DNA.

Vectors of the present invention for use in yeast will typically contain an origin of replication suitable for use in yeast and a selectable marker that is functional in yeast. Examples of yeast vectors useful in the present invention include, but are not limited to, Yeast Integrating plasmids (e.g., YIp5) and Yeast Replicating plasmids (the YRp and YEp series plasmids), Yeast Centromere plasmids (the YCp series plasmids), Yeast Artificial Chromosomes (YACs) which are based on yeast linear plasmids, denoted YLp, pGPD-2, 2μ plasmids and derivatives thereof, and improved shuttle vectors such as those described in Gietz et al., Gene, 74: 527–34 (1988) (YIplac, YEplac and YCplac).

Mammalian vectors useful for recombinant expression may include a viral origin, such as the SV40 origin (for replication in cell lines expressing the large T-antigen, such as COS1 and COS7 cells), the papillomavirus origin, or the EBV origin for long term episomal replication (for use, e.g., in 293-EBNA cells, which constitutively express the EBV EBNA-1 gene product and adenovirus E1A). Expression in mammalian cells can be achieved using a variety of plasmids, including, but not limited to, pSV2, pBC12BI, and p91023, pCDNA vectors as well as lytic virus vectors (e.g., vaccinia virus, adeno virus, and baculovirus), episomal virus vectors (e.g., bovine papillomavirus), and retroviral vectors (e.g., murine retroviruses). Useful vectors for insect cells include baculoviral vectors and pVL941.

Selection of an appropriate promoter to direct mRNA transcription and construction of expression vectors are well known. In general, however, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating codon at the beginning and a termination codon appropriately positioned at the end of the polypeptide to be translated.

Examples of useful promoters for prokaryotes include, but are not limited to phage promoters such as phage lambda pL promoter, the trc promoter, a hybrid derived from the trp and lac promoters, the bacteriophage T7 promoter, the TAC or TRC system, the major operator and promoter regions of phage lambda, the control regions of fd coat protein, snpA promoter, melC promotor, ermE* promoter or the araBAD operon. Examples of useful promoters for yeast include, but are not limited to, the CYC1 promoter, the GAL1 promoter, the GAL10 promoter, ADH1 promoter, the promoters of the yeast α-mating system, and the GPD promoter. Examples of promoters routinely used in mammalian expression vectors include, but are not limited to, the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous Sarcoma Virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

Vectors comprising the polynucleotides can be introduced into host cells using any number of well known techniques including infection, transduction, transfection, transvection and transformation. The polynucleotides may be introduced into a host alone or with additional polynucleotides encoding, for example, a selectable marker or ferredoxin reductase. In a preferred embodiment of the present invention the polynucleotide for epothilone B hydroxylase and ferredoxin are introduced into the host cell. Host cells for the various expression constructs are well known, and those of skill can routinely select a host cell for expressing the epothilone B hydroxylase and/or ferredoxin in accordance with this aspect of the present invention. Examples of mammalian expression systems useful in the present invention include, but are not limited to, the C127, 3T3, CHO, HeLa, human kidney 293 and BHK cell lines, and the COS-7 line of monkey kidney fibroblasts.

Alternatively, as exemplified herein, epothilone B hydroxylase and ferredoxin can be expressed recombinantly in microorganisms.

Accordingly, another aspect of the present invention relates to recombinantly produced microorganisms which express epothilone B hydroxylase alone or in conjunction with the ferredoxin and which are capable of hydroxylating a compound, and in particular an epothilone, having a terminal alkyl group to produce ones having a terminal hydroxyalkyl group. The recombinantly produced microorganisms are produced by transforming cells such as bacterial cells with a plasmid comprising a nucleic acid sequence encoding epothilone B hydroxylase. In a preferred embodiment, the cells are transformed with a plasmid comprising a nucleic acid encoding epothilone B hydroxylase or mutants or variants thereof as well as the nucleic acid sequence encoding ferredoxin located downstream of the epothilone B hydroxylase gene. Examples of microorganisms which can be transformed with these plasmids to produce the recombinant microorganisms of the present invention include, but are not limited, Escherichia coli, Bacillus megaterium, Amycolatopsis orientalis, Sorangium cellulosum, Rhodococcus erythropolis, and Streptomyces species such as Streptomyces lividans, Streptomyces virginiae, Streptomyces venezuelae, Streptomyces albus, Streptomyces coelicolor, Streptomyces rimosus and Streptomyces griseus.

The recombinantly produced microorganisms of the present invention are useful in microbial processes or methods for production of compounds, and in particular epothilones, containing a terminal hydroxyalkyl group. In general, the hydroxyalkyl-bearing product can be produced by culturing the recombinantly produced microorganism or enzyme derived therefrom, capable of selectively hydroxylating a terminal carbon or alkyl, in the presence of a suitable substrate in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, under submerged aerobic conditions.

Suitable epothilones employed as substrate for the method of the present invention may be any such compound having a terminal carbon or terminal alkyl group capable of undergoing the enzymatic hydroxylation of the present invention. The starting material, or substrate, can be isolated from natural sources, such as Sorangium cellulosum, or they can be synthetically formed epothilones. Other substrates having a terminal carbon or terminal alkyl group capable of undergoing an enzymatic hydroxylation can be employed by the methods herein. For example, compactin can be used as a substrate, which upon hydroxylation forms the compound pravastatin. Methods for hydroxylating compactin to pravastatin via an Actinomadura strain are set forth in U.S. Pat. No. 5,942,423 and U.S. Pat. No. 6,274,360.

For example, using the recombinant microorganisms of the present invention at least one epothilone can be prepared as described in WO 00/39276, U.S. Ser. No. 09/468,854, filed Dec. 21, 1999, the text of which is incorporated herein as if set forth at length. An epothilone of the following Formula I HO—CH₂-(A₁)_(n)-(Q)_(m)-(A₂)_(o)-E  (I) where

A₁ and A₂ are independently selected from the group of optionally substituted C₁–C₃ alkyl and alkenyl;

Q is an optionally substituted ring system containing one to three rings and at least one carbon to carbon double bond in at least one ring;

n, m, and o are integers selected from the group consisting of zero and 1, where at least one of m or n or o is 1; and

E is an epothilone core; can be prepared.

This method comprises the steps of contacting at least one epothilone of the following formula II CH₃-(A₁)_(n)-(Q)_(m)-(A₂)_(o)-E  (II)

where A₁, Q, A₂, E, n, m, and o are defined as above;

with a recombinantly produced microorganism, or an enzyme derived therefrom, which is capable of selectively catalyzing the hydroxylation of formula II, and effecting said hydroxylation.

In a preferred embodiment, the starting material is epothilone B. Epothilone B can be obtained from the fermentation of Sorangium cellulosum So ce90, as described in DE 41 38 042 and WO 93/10121. The strain has been deposited at the Deutsche Sammlung von Mikroorganismen (German Collection of Microorganisms) (DSM) under No. 6773. The process of fermentation is also described in Hofle, G., et al., Angew. Chem. Int. Ed. Engl., Vol 35, No. 13/14, 1567–1569 (1996). Epothilone B can also be obtained by chemical means, such as those disclosed by Meng, D., et al., J. Am. Chem. Soc., Vol. 119, No. 42, 10073–10092 (1996); Nicolaou, K., et al., J. Am. Chem. Soc., Vol. 119, No. 34, 7974–7991 (1997) and Schinzer, D., et al., Chem. Eur. J., Vol. 5, No. 9, 2483–2491 (1999).

Growth of the recombinantly produced microorganism selected for use in the process may be achieved by one of ordinary skill in the art by the use of appropriate nutrient medium. Appropriate media for the growing of the recombinantly produced microorganisms include those that provide nutrients necessary for the growth of microbial cells. See, for example, T. Nagodawithana and J. M. Wasileski, Chapter 2: “Media Design for Industrial Fermentations,” Nutritional Requirements of Commercially Important Microorganism, edited by T. W. Nagodawithana and G. Reed, Esteekay Associates, Inc., Milwaukee, Wis., 18–45 (1998); T. L. Miller and B. W. Churchill, Chapter 10: “Substrates for Large-Scale Fermentations,” Manual of Industrial Microbiology and Biotechnology, edited by A. L. Demain and N. A. Solomon, American Society for Microbiology, Washington, D.C., 122–136 (1986). A typical medium for growth includes necessary carbon sources, nitrogen sources, and trace elements. Inducers may also be added to the medium. The term inducer as used herein, includes any compound enhancing formation of the desired enzymatic activity within the recombinantly produced microbial cell. Typical inducers as used herein may include solvents used to dissolve substrates, such as dimethyl sulfoxide, dimethyl formamide, dioxane, ethanol and acetone. Further, some substrates, such as epothilone B, may also be considered to be inducers.

Carbon sources may include sugars such as glucose, fructose, galactose, maltose, sucrose, mannitol, sorbital, glycerol starch and the like; organic acids such as sodium acetate, sodium citrate, and the like; and alcohols such as ethanol, propanol and the like. Preferred carbon sources include, but are not limited to, glucose, fructose, sucrose, glycerol and starch.

Nitrogen sources may include an N-Z amine A, corn steeped liquor, soybean meal, beef extract, yeast extract, tryptone, peptone, cottonseed meal, peanut meal, amino acids such as sodium glutamate and the like, sodium nitrate, ammonium sulfate and the like.

Trace elements may include magnesium, manganese, calcium, cobalt, nickel, iron, sodium and potassium salts. Phosphates may also be added in trace or preferably, greater than trace amounts.

The medium employed for the fermentation may include more than one carbon or nitrogen source or other nutrient.

For growth of the recombinantly produced microorganisms and/or hydroxylation according to the method of the present invention, the pH of the medium is preferably from about 5 to about 8 and the temperature is from about 14° C. to about 37° C., preferably the temperature is 28° C. The duration of the reaction is 1 to 100 hours, preferably 8 to 72 hours.

The medium is incubated for a period of time necessary to complete the biotransformation as monitored by high performance liquid chromatography (HPLC). Typically, the period of time needed to complete the transformation is twelve to one hundred hours and preferably about 72 hours after the addition of the substrate. The medium is placed on a rotary shaker (New Brunswick Scientific Innova 5000) operating at 150 to 300 rpm and preferably about 250 rpm with a throw of 2 inches.

The hydroxyalkyl-bearing product can be recovered from the fermentation broth by conventional means that are commonly used for the recovery of other known biologically active substances. Examples of such recovery means include, but are not limited to, isolation and purification by extraction with a conventional solvent, such as ethyl acetate and the like; by pH adjustment; by treatment with a conventional resin, for example, by treatment with an anion or cation exchange resin or a non-ionic adsorption resin; by treatment with a conventional adsorbent, for example, by distillation, by crystallization; or by recrystallization, and the like.

The extract obtained above from the biotransformation reaction mixture can be further isolated and purified by column chromatography and analytical thin layer chromatography.

The ability of a recombinantly produced microorganism of the present invention to biotransform an epothilone having a terminal alkyl group to an epothilone having a terminal hydroxyalkyl group was demonstrated. In these experiments, a culture comprising a Streptomyces lividans clone containing a plasmid with the ebh gene as described in more detail in Example 11 was incubated with an epothilone B suspension for 3 days at 30° with agitation. A sample of the incubate was extracted with an equal volume of 25% methanol: 75% n-butanol, vortexed and allowed to settle for 5 minutes. Two hundred μl of the organic phase was transferred to an HPLC vial and analyzed by HPLC/MS (Example 12). A product peak of epothilone F eluted at a retention time of 15.9 minutes and had a protonated molecular weight of 524. The epothilone B substrate eluted at 19.0 minutes and had a protonated molecular weight of 508. The peak retention times and molecular weights were confirmed using known standards.

Rates of biotransformation of epothilone B by cells expressing ebh were also compared to rates of biotransformation by ebh mutants. Cells expressing ebh comprised a frozen spore preparation of. S. lividans (pANT849-ebh). Cells expressing mutants comprises frozen spore preparations of S. lividans (pANT849-ebh10-53) and S. lividans (pANT849-ebh24-16). A frozen spore preparation of S. lividans TK24 was used as the control. The cells were pre-incubated for several days at 30° C. Following this pre-incubation, epothilone B in 100% EtOH was added to each culture to a final concentration of 0.05% weight/volume. Samples were then taken at 0, 24, 48 and 72 hours with the exception of the S. lividans (pANT849-ebh24-16) culture, in which the epothilone B had been completely converted to epothilone F at 48 hours. The samples were analyzed by HPLC. The results are calculated as a percentage of the epothilone B at time 0 hours.

pANT849- Time (hours) TK24 pANT849-ebh pANT849-ebh10-53 ebh24-16 0 100% 100% 100% 100% 24  99%  78%  69%  56% 48  87%  19%  39%  0% 72  87%  0%  3% —

pANT849- Time (hours) TK24 pANT849-ebh pANT849-ebh10-53 ebh24-16 0 0%  0%  0%  0% 24 0%  4%  9% 23% 48 0% 21% 29% 52% 72 0% 14% 41% —

The ability of cells expressing ebh to biotransform compactin to pravastatin was also examined. In these experiments, frozen spore preparations of S. lividans (pANT849) or S. lividans (pANT849-ebh) were grown for several days at 30° C. Following the pre-incubation, an aliquot of each cell culture was transferred to a polypropylene culture tube, compactin was added to each culture tube, and the tubes were incubated for 24 hours, 30° C., 250 rpm. An aliquot of the culture broth was then extracted and compactin and pravastatin values relative to the control S. lividans (pANT849) culture were measured via HPLC.

Compactin and pravastatin as a percentage of starting compactin concentration:

S. lividans (pANT849) S. lividans (pANT849-ebh) Compactin 36% 11% Pravastatin 11% 53%

As discussed supra, mutant ebh25-1 (SEQ ID NO:30) exhibits altered substrate specificity and biotransformation of epothilone B by this mutant resulted in a product with a different HPLC elution time than epothilone B or epothilone F. A sample of this unknown was analyzed by LC-MS and was found to have a molecular weight of 523 (M.W.), consistent with a single hydroxylation of epothilone B. The structure of the biotransformation product was determined as 24-hydroxyl-epothilone B, based on MS and NMR data (compared with data of epothilone B):

Molecular Formula: C₂₇H₄₁NO₇S Molecular Weight: 523 Mass Spectrum: ES+ (m/z): 524([M+H]⁺), 506. LC/MS/MS: +ESI (m/z): 524, 506, 476, 436, 320 HRMS: Calculated for [M+H]⁺: 524.2682; Found: 524.2701 HPLC (Rt) 7.3 minutes (on the analytical HPLC system) LC/NMR Observed Chemical Shifts Varian AS-600 (Proton: 599.624 MHz), Solvent D₂O/CD₃CN (δ 1.94): ˜4/6 Proton: δ7.30 (s, 1H), 6.43 (s, 1H), 5.30 (m, 1H), 4.35 (m, 1H), 3.81 (m, 1H), 3.74 (m, 1H), 3.68 (m, 1H), 3.43 (m, 1H), 2.87 (m, 1H), 2.66 (s, 3H), 2.40 (m, 2H), 1.58 (b, 1H), 1.48 (b, 1H), 1.35 (m, 3H), 1.18 (s, 3H), 1.13 (s, 3H), 0.87 (m, 6H) *Peaks between 1.8–2.1 ppm were not observed due to solvent suppression.

The proton chemical shift was assigned as follows:

Position Proton Pattern 1 — 2 2.40 m 3 4.35 m 4 — 5 — 6 3.43 m 7 3.68 m 8 1.58 m 9 1.35 b 10 1.48 b 10 1.35 b 11 SSP 12 — 13 2.87 m 14 SSP 15 5.30 m 16 — 17 6.43 s 18 — 19 7.30 s 20 — 21 2.66 s 22 1.18 s 23 0.87 m 24 3.81 m 24 3.74 m 25 0.87 m 26 1.13 s 27 SSP *SSP: no observed due to solvent suppression.

Accordingly, the compositions and methods of the present invention are useful in producing known compounds that are microtubule-stabilizing agents as well as new compounds comprising epothilone analogs such as 24-hydroxyl-epothilone B (Formula A) and pharmaceutically acceptable salts thereof expected to be useful as microtubule-stabilizing agents. The microtubule stabilizing agents produced using these compositions and methods are useful in the treatment of a variety of cancers and other proliferative diseases including, but not limited to, the following;

-   -   carcinoma, including that of the bladder, breast, colon, kidney,         liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin;         including squamous cell carcinoma;     -   hematopoietic tumors of lymphoid lineage, including leukemia,         acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell         lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins         lymphoma, hairy cell lymphoma and Burketts lymphoma;     -   hematopoietic tumors of myeloid lineage, including acute and         chronic myelogenous leukemias and promyelocytic leukemia;     -   tumors of mesenchymal origin, including fibrosarcoma and         rhabdomyoscarcoma;     -   other tumors, including melanoma, seminoma, tetratocarcinoma,         neuroblastoma and glioma;     -   tumors of the central and peripheral nervous system, including         astrocytoma, neuroblastoma, glioma, and schwannomas;     -   tumors of mesenchymal origin, including fibrosarcoma,         rhabdomyosarcoma, and osteosarcoma; and     -   other tumors, including melanoma, xenoderma pigmentosum,         keratoactanthoma, seminoma, thyroid follicular cancer and         teratocarcinoma.

Microtubule stabilizing agents produced using the compositions and methods of the present invention will also inhibit angiogenesis, thereby affecting the growth of tumors and providing treatment of tumors and tumor-related disorders. Such anti-angiogenesis properties of these compounds will also be useful in the treatment of other conditions responsive to anti-angiogenesis agents including, but not limited to, certain forms of blindness related to retinal vascularization, arthritis, especially inflammatory arthritis, multiple sclerosis, restinosis and psoriasis.

Microtubule stabilizing agents produced using the compositions and methods of the present invention will induce or inhibit apoptosis, a physiological cell death process critical for normal development and homeostasis. Alterations of apoptotic pathways contribute to the pathogenesis of a variety of human diseases. Compounds of the present invention such as those set forth in formula I and II and Formula A, as modulators of apoptosis, will be useful in the treatment of a variety of human diseases with aberrations in apoptosis including, but not limited to, cancer and precancerous lesions, immune response related diseases, viral infections, degenerative diseases of the musculoskeletal system and kidney disease.

Without wishing to be bound to any mechanism or morphology, microtubule stabilizing agents produced using the compositions and methods of the present invention may also be used to treat conditions other than cancer or other proliferative diseases. Such conditions include, but are not limited to viral infections such as herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus; autoimmune diseases such as systemic lupus erythematosus, immune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel diseases and autoimmune diabetes mellitus; neurodegenerative disorders such as Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration; AIDS; myelodysplastic syndromes; aplastic anemia; ischemic injury associated myocardial infarctions; stroke and reperfusion injury; restenosis; arrhythmia; atherosclerosis; toxin-induced or alcohol induced liver diseases; hematological diseases such as chronic anemia and aplastic anemia; degenerative diseases of the musculoskeletal system such as osteoporosis and arthritis; aspirin-sensitive rhinosinusitis; cystic fibrosis; multiple sclerosis; kidney diseases; and cancer pain.

The following nonlimiting examples are provided to further illustrate the present invention.

EXAMPLES Example 1 Reagents

R2 Medium was prepared as follows:

A solution containing sucrose (103 grams), K₂SO₄ (0.25 grams) MgCl₂.6H₂O (10.12 grams), glucose (10 grams), Difco Casaminoacids (0.1 grams) and distilled water (800 ml) was prepared. Eighty ml of this solution was then poured into a 200 ml screw capped bottle containing 2.2 grams Difco Bacto agar. The bottle was capped and autoclaved. At time of use, the medium was remelted and the following autoclaved solutions were added in the order listed:

1 ml KH₂PO₄ (0.5%)

8 ml CaCl₂.2H₂O (3.68%)

1.5 ml L-proline (20%)

10 ml TES buffer (5.73%, adjusted to pH 7.2)

0.2 ml Trace element solution containing ZnCl₂ (40 mg), FeCl₃.6H₂O (200 mg), CuCl₂.2H₂O (10 mg), MnCl₂.4H₂O (10 mg), Na₂B₄O₇.10H₂O (10 mg), and (NH₄)₆Mo₇O₂₄.H₂O

0.5 ml NaOH (1N)(sterilization not required)

0.5 ml Required growth factors for auxotrophs (Histidine (50 μg/ml); Cysteine (37 μg/ml); adenine, guanine, thymidine and uracil (7.5 μg/ml); and Vitamins (0.5 μg/ml).

R2YE medium was prepared in the same fashion as R2 medium. However, 5 ml of Difco yeast extract (10%) was added to each 100 ml flask at time of use.

P (protoplast) buffer was prepared as follows:

A basal solution made up of the following was prepared:

Sucrose (103 grams)

K₂SO₄ (0.25 grams)

MgCl₂.6H₂O (2.02 grams)

Trace Element Solution as described for R2 medium (2 ml)

Distilled water to 800 ml

Eighty ml aliquots of the basal solution were then dispensed and autoclaved. Before use, the following was added to each flask in the order listed:

1 ml KH₂PO₄ (0.5%)

10 ml CaCl₂.2H₂O (3.68%)

TES buffer (5.75%, adjusted to pH 7.2)

T (transformation) buffer was prepared by mixing the following sterile solutions:

25 ml Sucrose (10.3%)

75 ml distilled water

1 ml Trace Element Solution as described for R2 medium

1 ml K₂SO₄ (2.5%)

The following are then added to 9.3 mls of this solution:

0.2 ml CaCl₂ (5M)

0.5 ml Tris maleic acid buffer prepared from 1 M solution of Tris adjusted to pH 8.0 by adding maleic acid.

For use, 3 parts by volume of the above solution are added to 1 part by weight of PEG 1000, previously sterilized by autoclaving.

L (lysis) buffer was prepared by mixing the following sterile solutions:

100 ml Sucrose (10.3%)

10 ml TES buffer (5.73%, adjusted to pH 7.2)

1 ml K₂SO₄ (2.5%)

1 ml Trace Element Solution as described for R2 medium

1 ml KH₂PO₄ (0.5%)

0.1 ml MgCl₂.6H₂O (2.5 M)

1 ml CaCl₂ (0.25 M)

CRM Medium

A solution containing the following components was prepared in 1 liter of dH₂O: glucose (10 grams), sucrose (103 grams), MgCl₂.6H₂O (10.12 grams), BBL™ trypticase soy broth (15 grams) (Becton Dickinson Microbiology Systems, Sparks, Md., USA), and BBL™ yeast extract (5 grams) (Becton Dickinson Microbiology Systems). The solution was autoclaved for 30 minutes. Thiostrepton was added to a concentration of 10 μg/ml for cultures propagated with plasmids.

Electroporation Buffer

A solution containing 30% (wt/vol) PEG 1000, 10% glycerol, and 6.5% sucrose was prepared in dH₂O. The solution was sterilized by vacuum filtration through a 0.22 μm cellulose acetate filter.

Example 2 Extraction of Chromosomal DNA from Strain SC15847

Genomic DNA was isolated from an Amycolatopsis orientalis soil isolate strain designation SC15847 (ATCC PT-1043) using a guanidine-detergent lysis method, DNAzol reagent (Invitrogen, Carlsbad, Calif., USA). The SC15847 culture was grown 24 hours at 28° C. in F7 medium (glucose 2.2%, yeast extract 1.0%, malt extract 1.0%, peptone 0.1%, pH 7.0). Twenty ml of culture was harvested by centrifugation and resuspended in 20 ml of DNAzol, mixed by pipetting and centrifuged 10 minutes in the Beckman TJ6 centrifuge. Ten ml of 100% ethanol was added, inverted several times and stored at room temperature 3 minutes. The DNA was spooled on a glass pipette washed in 100% ethanol and allowed to air dry 10 minutes. The pellet was resuspended in 500 μl of 8 mM NaOH and once dissolved it was neutralized with 30 μl of 1M HEPES pH7.2.

Example 3 PCR Reactions

PCR reactions were prepared in a volume of 50 μl, containing 200–500 ng of genomic DNA or 1.0 μl of the cDNA, a forward and reverse primer, and the forward primer being either P450-1⁺ (SEQ ID NO:23) or P450-1a⁺ (SEQ ID NO:24) or P450-2⁺ (SEQ ID NO:25) and the reverse primer P450-3⁻ (SEQ ID NO:27) or P450-2⁻ (SEQ ID NO:26). All primers were added to a final concentration of 1.4–2.0 μM. The PCR reaction was prepared with 1 μl of Taq enzyme (2.5 units) (Stratagene), 5 μl of Taq buffer and 4 μl of 2.5 mM of dNTPs with dH₂O to 50 μl. The cycling reactions were performed on a Geneamp® PCR system with the following protocol: 95° C. for 5 minutes, 5 cycles [95° C. 30 seconds, 37° C. 15 seconds (30% ramp), 72° C. 30 seconds], 35 cycles (94° C. 30 seconds, 65° C. 15 seconds, 72° C. 30 seconds), 72° C. 7 minutes. The expected sizes for the reactions are 340 bp for the P450-1⁺ (SEQ ID NO:23) or P450-1a⁺ (SEQ ID NO:24) and P450-3⁻ (SEQ ID NO:27) primer pairs, 240 bp for the P450-1⁺ (SEQ ID NO:23) and P450-2⁻ (SEQ ID NO:26) primer pairs and 130 bp for the P450-2⁺ (SEQ ID NO:25) and P450-3⁻ (SEQ ID NO:27) primer pairs.

Example 4 Cloning of Epothilone B Hydroxylase and Ferredoxin Genes

Twenty μg of SC15847 genomic DNA was digested with BglII restriction enzyme for 6 hours at 37° C. A 30k nanosep column (Gelman Sciences, Ann Arbor, Mich., USA) was used to concentrate the DNA and remove the enzyme and buffer. The reactions were concentrated to 40 μl and washed with 200 μl of TE. The digestion products were then separated a 0.7% agarose gel and genomic DNA in the range of 12˜15 kb was excised from the gel and purified using the Qiagen gel extraction method. The genomic DNA was then ligated to plasmid pWB19N (U.S. Pat. No. 5,516,679), which had been digested with BamHI and dephosphorylated using the SAP I enzyme (Roche Molecular Biochemicals, Indianapolis, Ind., catalog#1 758 250). Ligation reactions were performed in a 15 μl volume with 1 U of T4 DNA ligase (Invitrogen) for 1 hour at room temperature. One μl of the ligation was transformed to 100 μl of chemically competent DH10B cells (Invitrogen) and 100 μl plated to five LB agar plates with 30 μg/ml of neomycin, 37° C. overnight.

Five nylon membrane circles (Roche Molecular Biochemicals, Indianapolis, Ind.) were numbered and marked for orientation. The membranes were placed on the plates 2 minutes and then allowed to dry for 5 minutes. The membranes were then placed on Whatman filter disks saturated with 10% SDS for 5 minutes, 0.5N NaOH with 1.5 M NaCl for 5 minutes, 1.5 M NaCl with 1.0 M Tris pH 8.0 for 5 minutes, and 15 minutes on 2×SSC. The filters were hybridized as described previously for the Southern hybridization. Hybridizing colonies were picked to 2 ml of TB with 30 μg/ml neomycin and grown overnight at 37° C. Plasmid DNA was isolated using a miniprep column procedure (Mo Bio). This plasmid was named NPB29-1.

Example 5 DNA Sequencing and Analysis

The cloned PCR products were sequenced using fluorescent-dye-labeled terminator cycle sequencing, Big-Dye sequencing kit (Applied Biosystems, Foster city, Calif., USA) and were analyzed using laser-induced fluorescence capillary electrophoresis, ABI Prism 310 sequencer (Applied Biosystems).

Example 6 Extraction of Total RNA

Total RNA was isolated from the SC15847 culture using a modification of the Chomczynski and Sacchi method with a mono-phasic solution of phenol and guanidine isothiocyanate, Trizol reagent (Invitrogen). Five ml of an SC15847 frozen stock culture was thawed and used to inoculate 100 ml of F7 media in a 500 ml Erlenmeyer flask. The culture was grown in a shaker incubator at 230 rpm, 30° C. for 20 hours to an optical density at 600 nm (OD₆₀₀) of 9.0. The culture was placed in a 16° C. shaker incubator at 230 rpm for 20 minutes. Fifty-five milligrams of epothilone B was dissolved in 1 ml of 100% ethanol and added to the culture. A second ml of ethanol was used to rinse the residual epothilone B from the tube and added to the culture. The culture was incubated at 16° C., 230 rpm for 30 hours. Thirty ml of the culture was transferred to a 50 ml tube, 150 mg of lysozyme was added to the culture and the culture was incubated 5 minutes at room temperature. Ten ml of the culture was placed in a 50 ml Falcon tube and centrifuged 5 minutes, 4° C. in a TJ6 centrifuge. Two ml of chloroform was added and the tube was mixed vigorously for 15 seconds. The tube was incubated 2 minutes at room temperature and centrifuged 10 minutes, top speed in the TJ6 centrifuge. The aqueous layer was transferred to a fresh tube and 2.5 ml of isopropanol was added to precipitate the RNA. The tube was incubated 10 minutes at room temperature and centrifuged 10 minutes, 4° C. The supernatant was removed, the pellet was rinsed with 70% ethanol and dried briefly under vacuum. The pellet was resuspended in 150 μl of RNase-free dH₂O. Fifty μl of 7.5M LiCl was added to the RNA and incubated at −20° C. for 30 minutes. The RNA was pelleted by centrifugation 10 minutes, 4° C. in a microcentrifuge. The pellet was rinsed with 200 μl of 70% ethanol, dried briefly under vacuum and resuspended in 150 μl of RNase free dH₂O.

The RNA was treated with DNaseI (Ambion, Austin, Tex., USA). Twenty-five μl of total RNA (5.3 μg/μl), 2.5 μl of DNaseI buffer, 1.0 μl of DNase I added and incubated at 37° C. for 25 minutes. Five μl of DNase I inactivation buffer added, incubated 2 minutes, centrifuged 1 minute, the supernatant was transferred to a fresh tube.

Example 7 cDNA Synthesis

cDNA was synthesized from the total RNA using the Superscript II enzyme (Invitrogen). The reaction was prepared with 1 μl of total RNA (5.3 μg/μl), 9 μl of dH₂O, 1 μl of dNTP mix (10 mM), and 1 μl of random hexamers. The reaction was incubated at 65° C. for 5 minutes then placed on ice. The following components were then added: 4 μl of 1^(st) strand buffer, 1 μl of RNase Inhibitor, 2.0 μl of 0.1 M DTT, and 1 μl of Superscript II enzyme. The reaction was incubated at room temperature 10 minutes, 42° C. for 50 minutes and 70° C. for 15 minutes. One μl of RNaseH was added and incubated 20 minutes at 37° C., 15 minutes at 70° C. and stored at 4° C.

Example 8 DNA Labeling

The PCR conditions used to amplify the P450 specific products from genomic DNA and cDNA were used to amplify the insert of plasmid pCRscript-29. Plasmid pCRscript-29 contains a 340 bp PCR fragment amplified from SC15847 genomic DNA using primers P450 1⁺ (SEQ ID NO:23) and P450 3⁻ (SEQ ID NO:27). Two μl of the plasmid prep was used as a template, with a total of 25 cycles. The amplified product was gel purified using the Qiaquick gel extraction system (Qiagen). The extracted DNA was ethanol precipitated and resuspended in 5 μl of TE, the yield was estimated to be 500 ng. This fragment was labeled with digoxigenin using the chem link labeling reagent (Roche Molecular Biochemicals, Indianapolis, Ind. catalog #1 836 463). Five μl of the PCR product was mixed with 0.5 μl of Dig-chem link and dH₂O added to 20 μl. The reaction was incubated 30 minutes at 85° C. and 5 μl of stop solution added. The probe concentration was estimated at 20 ng/μl.

Example 9 Southern DNA Hybridization

Ten μl of genomic DNA (0.5 μg/l) was digested with BamHI, BglII, EcoRI, HindIII or NotI and separated at 12 volts for 16 hours. The gel was depurinated 10 minutes in 0.25 N HCl and transferred by vacuum to a nylon membrane (Roche Molecular Biochemicals) in 0.4 N NaOH 5″ Hg, 90 minutes using a vacuum blotter (Bio-Rad Laboratories, Inc. Hercules, Calif., USA catalog # 165-5000). The membrane was rinsed in 1 M ammonium acetate and UV-crosslinked using the Stratalinker UV Crosslinker (Stratagene). The membrane was rinsed in 2×SSC and stored at room temperature.

The membrane was prehybridized 1 hour at 42° C. in 20 ml of Dig Easy Hyb buffer (Roche Molecular Biochemicals). The probe was denatured 10 minutes at 65° C. and then placed on ice. Five ml of probe in Dig-Easy Hyb at an approximate concentration on 20 ng/ml was incubated with the membrane at 42° C. overnight. The membrane was washed 2 times in 2×SCC with 0.1% SDS at room temperature, then 2 times in 0.5×SSC with 0.1% SDS at 65° C. The membrane was equilibrated in Genius buffer 1 (10 mM maleic acid, 15 mM NaCl; pH 7.5; 0.3% v/v Tween 20) (Roche Molecular Biochemicals, Indianapolis, Ind.) for 2 minutes, then incubated with 2% blocking solution (2% Blocking reagent in Genius Buffer 1)(Roche Molecular Biochemicals Indianapolis, Ind.) for 1 hour at room temperature. The membrane was incubated with a 1:20,000 dilution of anti-dig antibody in 50 ml of blocking solution for 30 minutes. The membrane was washed 2 times, 15 minutes each in 50 ml of Genius buffer 1. The membrane was equilibrated for two minutes in Genius Buffer 3 (10 mM Tris-HCl, 10 mM NaCl; pH 9.5). One ml of a 1:100 dilution of CSPD (disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo[3.3.1.1^(3,7)]decan}-4-yl)phenyl phosphate) (Roche Molecular Biochemicals) in Genius buffer 3 was added to the membrane and incubated 5 minutes at room temperature, then placed at 37° C. for 15 minutes. The membrane was exposed to Biomax ML film (Kodak, Rochester, N.Y., USA) for 1 hour.

Example 10 E. coli Transformation

Competent cells were purchased from Invitrogen. E. coli strain DH10B was used as a host for genomic cloning. The chemically competent cells were thawed on ice and 100 μl aliquoted to a 17×100-mm polypropylene tube on ice. One μl of the ligation mixture was added to the cells and incubated on ice for 30 minutes. The cells were incubated at 42° C. for 45 seconds, then placed on ice 1–2 minutes. 0.9 ml pf SOC. medium (Invitrogen) was added and the cells were incubated one hour at 30–37° C. at 200–240 rpm. Cells were plated on a selective medium (Luria agar with neomycin or ampicillin at a concentration of 30 μg/ml or 100 μg/ml respectively).

Example 11 Transformation of Streptomyces lividans TK24

Plasmid pWB19N849 was constructed by digesting plasmid pWB19N with HindIII and treating with SAP I and digesting plasmid pANT849 (Keiser, et al., 2000, Practical Streptomyces Genetics, John Innes) with HindIII. The two linearized fragments were ligated 1 hour at room temperature with 1 U of T4 DNA ligase. One μl of the ligation reaction was used to transform XL-1 Blue electrocompetent cells (Stratagene). The recovered cells were plated to LB neomycin (30 μg/ml) overnight at 37° C. Colonies were picked to 2 ml of LB with 30 μg/ml neomycin and incubated overnight at 30° C. MoBio plasmid minipreps were performed on all cultures. Plasmids constructed from the ligation of pWB19N and pANT849 were determined by electrophoretic mobility on 0.7% agarose. The plasmid pWB19N849 was digested with HindIII and BglII to excise a 5.3 kb fragment equivalent to plasmid pANT849 digested with BglII and HindIII. This 5.3 kb fragment was purified on an agarose gel and extracted using the Qiaquick gel extraction system.

A 1.469 kb DNA fragment containing the epothilone B hydroxylase gene and the downstream ferredoxin gene was amplified using PCR. The 50 μl PCR reaction was composed of 5 μl of Taq buffer, 2.5 μl glycerol, 1 μl of 20 ng/μl NPB29-1 plasmid, 0.4 μl of 25 mM dNTPs, 1.0 μl each of primers NPB29-6F (SEQ ID NO:28) and NPB29-7R (SEQ ID NO:29) (5 pmole/μl), 38.1 μl of dH₂O and 0.5 μl of Taq enzyme (Stratagene). The reactions were performed on a Perkin Elmer 9700, 95° C. for 5 minutes, then 30 cycles (96° C. for 30 seconds, 60° C. 30 seconds, 72° C. for 2 minutes), and 72° C. for 7 minutes. The PCR product was purified using a Qiagen minielute column with the PCR cleanup procedure. The purified product was digested with BglII and HindIII and purified on a 0.7% agarose gel. A 1.469 kb band was excised from the gel and eluted using a Qiagen minielute column. Five μl of this PCR product was ligated with 2 μl of the BglII, HindIII digested pANT849 vector in a 10 μl ligation reaction. The reaction was incubated at room temperature for 24 hours and then transformed to S. lividans TK24 protoplasts.

Twenty ml of YEME media was inoculated with a frozen spore suspension of S. lividans TK24 and grown 48 hours in a 125 ml bi-indent flask. Protoplasts were prepared as described in Practical Streptomyces Genetics. The ligation reaction was mixed with protoplasts, then 500 μl of transformation buffer was added, followed immediately by 5 ml of P buffer. The transformation reactions were spun down 7 minutes at 2,750 rpm, resuspended in 100 μl of P buffer and plated to one R2YE plate. The plate was incubated at 28° C. for 20 hours then overlaid with 5 ml of LB 0.7% agar with 250 μg/ml thiostrepton. After 7 days colonies were picked to an R2YE grid plate with 50 μg/ml of thiostrepton. The colonies were grown an additional 5 days at 28° C., then stored at 4° C.

This recombinant microorganism has been deposited with the ATCC and designated PTA-4022.

Example 12 Transformation of Streptomyces rimosus

The procedure of Pigac and Schrempf Appl. Environ Microb., Vol. 61, No. 1, 352–356 (1995) was used to transform S. rimosus. S. rimosus strain R6 593 was cultivated in 20 ml of CRM medium at 30° C. on a rotary shaker (250 rpm). The cells were harvested at 24 hrs by centrifugation for 5 minutes, 5,000 rpm, 4° C., and resuspended in 20 ml of 10% sucrose, 4° C., and centrifuged for 5 minutes, 5,000 rpm, 4° C. The pellet was resuspended in 10 ml of 15% glycerol, 4° C. and centrifuged for 5 minutes, 5,000 rpm, 4° C. The pellet was resuspended in 2 ml of 15% glycerol, 4° C. with 100 μg/ml lysozyme and incubated at 37° C. for 30 minutes, centrifuged for 5 minutes, 5,000 rpm, 4° C. and resuspended in 2 ml of 15% glycerol, 4° C. The 15% glycerol wash was repeated once and the pellet was resuspended in 1 to 2 ml of Electroporation Buffer. The cells were stored at −80° C. in 50–200 μl aliquots.

The ligations were prepared as described for the S. lividans transformation. After the incubation of the ligation reaction, the volume was brought to 100 μl with dH₂O, NaCl was added to 0.3M, and the reaction extracted with an equal volume of 24:1:1 phenol:choroform isoamyl alcohol. Twenty μg of glycogen was added and the ligated DNA was precipitated with 2 volumes of 100% ethanol at −20° C. for 30 minutes. The DNA was pelleted 10 minutes in a microcentrifuge, washed once with 70% ethanol, dried 5 minutes in a speed-vac concentrator and resuspended in 5 μl of dH₂O.

One frozen aliquot of cells was thawed at room temperature and divided, 50 μl/tube for each DNA sample for electroporation. The cells were stored on ice until use. DNA in 1 to 2 μl of dH₂O was added and mixed. The cell and DNA mixture was transferred to a 2 mm gapped electrocuvette (Bio-Rad Laboratories, Richmond Calif. USA) that was pre-chilled on ice. The cells were electroporated at a setting of 2 kV (10 kV/cm), 25 μF, 400 Ω using a Gene Pulser™ (Bio-Rad Laboratories). The cells were diluted with 0.75 to 1.0 ml of CRM (0–4° C.), transferred to 15 ml culture tubes and incubated with agitation 3 hrs at 30° C. The cells were plated on trypticase soy broth agar plates with 10–30 μg/ml of thiostrepton.

Example 13 High Performance Liquid Chromatography

The liquid chromatography separation was performed using a Waters 2690 Separation Module system (Waters Corp., Milford, Mass., USA) and a column, 4.6×150 mm, filled with SymmetryShield RP₈, particle size 3.5 μm (Waters Corp., Milford, Mass., USA). The gradient mobile phase programming was used with a flow rate of 1.0 ml/minute. Eluent A was water/acetonitrile (20:1)+10 mM ammonium acetate. Eluent B was acetonitrile/water (20:1). The mobile phase was a linear gradient from 12% B to 28% B over 6 minutes and held isocratic at 28% B over 4 minutes. This was followed by a 28% B to 100% B linear gradient over 20 minutes and a linear gradient to 12% B over two minutes with a 3 minute hold at 12% B.

Example 14 Mass Spectrometry

The column effluent was introduced directly into the electrospray ion source of a ZMD mass spectrometer (Micromass, Manchester, UK). The instrument was calibrated-using Test Juice reference standard (Waters Corp, Milford, Mass., USA) and was delivered at a flow of 10 μl/minute from a syringe pump (Harvard Apparatus, Holliston, Mass., USA). The mass spectrometer was operated at a low mass resolution of 13.2 and a high mass resolution of 11.2. Spectra were acquired from using a scan range of m/z 100 to 600 at an acquisition rate of 10 spectra/second. The ionization technique employed was positive electrospray (ES). The sprayer voltage was kept at 2900 V and the cone voltage of the ion source was kept at a potential of 17 V.

Example 15 Use of the ebh Gene Sequence (SEQ ID NO:1) to Isolate Cytochrome P450 Genes from Other Microorganisms

Genomic DNA was isolated from a set of cultures (ATCC43491, ATCC14930, ATCC53630, ATCC53550, ATCC39444, ATCC43333, ATCC35165) using the DNAzol reagent. The DNA was used as a template for PCR reactions using primers designed to the sequence of the ebh gene. Three sets of primers were used for amplification; NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29), NPB29-16f (SEQ ID NO:50) and NPB29-17r (SEQ ID NO:51), and NPB29-19f (SEQ ID NO:52) and NPB29-20r (SEQ ID NO:53).

PCR reactions were prepared in a volume of 20 μl, containing 200–500 ng of genomic DNA and a forward and reverse primer. All primers were added to a final concentration of 1.4–2.0 μM. The PCR reaction was prepared with 0.2 μl of Advantage™ 2 Taq enzyme (BD Biosciences Clontech, Palo Alto, Calif., USA), 2 μl of Advantage™ 2 Taq buffer and 0.2 μl of 2.5 mM of dNTPs with dH₂O to 20 μl. The cycling reactions were performed on a Geneamp® 9700 PCR system or a Mastercycler® gradient (Eppendorf, Westbury, N.Y., USA) with the following protocol: 95° C. for 5 minutes, 35 cycles (96° C. 20 seconds, 54–69° C. 30 seconds, 72° C. 2 minutes), 72° C. for 7 minutes. The expected size of the PCR products is approximately 1469 bp for the NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29) primer pair, 1034 bp for the NPB29-16f (SEQ ID NO:50) and NPB29-17r (SEQ ID NO:51) primer pair and 1318 bp for the NPB29-19f (SEQ ID NO:52) and NPB29-20r (SEQ ID NO:53) primer pair. The PCR reactions were analyzed on 0.7% agarose gels. PCR products of the expected size were excised from the gel and purified using the Qiagen gel extraction method. The purified products were sequenced using the Big-Dye sequencing kit and analyzed using an AB1310 sequencer.

Example 16 Construction of Plasmid pPCRscript-ebh

A 1.469 kb DNA fragment containing the epothilone B hydroxylase gene and the downstream ferredoxin gene was amplified using PCR. The 50 μl PCR reaction was composed of 5 μl of Taq buffer, 2.5 μl glycerol, 1 μl of 20 ng/μl NPB29-1 plasmid, 0.4 μl of 25 mM dNTPs, 1.0 μl each of primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29) (5 pmole/μl), 38.1 μl of dH₂O and 0.5 μl of Taq enzyme (Stratagene). The reactions were performed on a Geneamp® 9700 PCR system, with the following conditions; 95° C. for 5 minutes, then 30 cycles (96° C. for 30 seconds, 60° C. 30 seconds, 72° C. for 2 minutes), and 72° C. for 7 minutes. The PCR product was purified using a Qiagen Qiaquick column with the PCR cleanup procedure. The purified product was digested with BglII and HindIII and purified on a 0.7% agarose gel. A 1.469 kb band was excised from the gel and eluted using a Qiagen Qiaquick gel extraction procedure. The fragments were then cloned into the pPCRscript Amp vector using the PCRscript Amp cloning kit. Colonies containing inserts were picked to 1–2 ml of LB (Luria Broth) with 100 μg/ml ampicillin, 30–37° C., 16–24 hours, 230–300 rpm. Plasmid isolation was performed using the Mo Bio miniplasmid prep kit. The sequence of the insert was confirmed by cycle sequencing with the Big-Dye sequencing kit. This plasmid was named pPCRscript-ebh.

Example 17 Mutagenesis of the ebh Gene for Improved Yield or Altered Specificity

The Quikchange® XL Site-Directed Mutagenesis Kit and the Quikchange® Multi Site-Directed Mutagenesis kit, both from Stratagene were used to introduce mutations in the coding region of the ebh gene. Both of these methods employ DNA primers 35–45 bases in length containing the desired mutation (SEQ ID NO:54–59 and 70), a methylated circular plasmid template and PfuTurbo® DNA Polymerase (U.S. Pat. Nos. 5,545,552 and 5,866,395 and 5,948,663) to generate copies of the plasmid template incorporating the mutation carried on the mutagenic primers. Subsequent digestion of the reaction with the restriction endonuclease enzyme DpnI, selectively digests the methylated plasmid template, but leaves the non-methylated mutated plasmid intact. The manufacturer's instructions were followed for all procedures with the exception of the DpnI digestion step in which the incubation time was increased from 1 hr to 3 hrs. The pPCRscript-ebh vector was used as the template for mutagenesis.

One to two μl of the reaction was transformed to either XL1-Blue® electrocompetent or XL10-Gold® ultracompetent cells (Stratagene). Cells were plated to a density of greater than 100 colonies per plate on LA (Luria Agar) 100 μg/ml ampicillin plates, and incubated 24–48 hrs at 30–37° C. The entire plate was resuspended in 5 ml of LB containing 100 μg/ml ampicillin. Plasmid was isolated directly from the resuspended cells by centrifuging the cells and then purifying the plasmid using the Mo Bio miniprep procedure. This plasmid was then used as a template for PCR with primers NPB29-6f (SEQ ID NO:28) and NPB29-7r (SEQ ID NO:29) to amplify a mutated expression cassette. Digestion of the 1.469 kb PCR product with the restriction enzymes BglII and HindIII was used to prepare this fragment for ligation to vector pANT849 also digested with BglII and HindIII. Alternatively, the resuspended cells were used to inoculate 20–50 ml of LB containing 100 μg/ml ampicillin and grown 18–24 hrs at 30–37° C. Qiagen midi-preps were performed on the cultures to isolate plasmid DNA containing the desired mutation. Digestion with the restriction enzymes BglII and HindIII was used to excise the mutated expression cassette for ligation to BglII and HindIII digested plasmid pANT849. Screening of mutants was performed in S. lividans or S. rimosus as described.

Alternatively, the method of Leung et al., Technique—A Journal of Methods in Cell and Molecular Biology, Vol. 1, No. 1, 11–15 (1989) was used to generate random mutation libraries of the ebh gene. Manganese and/or reduced dATP concentration is used to control the mutagenesis frequency of the Taq polymerase. The plasmid pCRscript-ebh was digested with NotI to linearize the plasmid. The Polymerase buffer was prepared with 0.166 M (NH₄)₂SO₄, 0.67M Tris-HCl pH 8.8, 61 mM MgCl₂, 67 μM EDTA pH8.0, 1.7 mg/ml Bovine Serum Albumin). The PCR reaction was prepared with 10 μl of Not I digested pCRscript-ebh (0.1 ng/μl), 10 μl of polymerase buffer, 1.0 μl of 1M β-mercaptoethanol, 10.0 μl of DMSO, 1.0 μl of NPB29-6f (SEQ ID NO:28) primer (100 pmole/μl), 1.0 μl of NPB29-7r (SEQ ID NO:29) primer (100 pmole/μl), 10 μl of 5 mM MnCl₂, 10.0 μl 10 mM dGTP, 10.0 μl 2 mM dATP, 10 mM dTTp, 10.0 μl Taq polymerase. dH₂O was added to 100 μl. Reactions were also prepared as described above but without MnCl₂. The cycling reactions were performed a GeneAmp® PCR system with the following protocol: 95° C. for 1 minute, 55° C. for 30 seconds, 72° C. for 4 minutes), 72° C. for 7 minutes. The PCR reactions were separated on an agarose gel using a Qiagen spin column. The fragments were then digested with BglII and HindIII and purified using a Qiagen spin column. The purified fragments were then ligated to BglII and HindIII digested pANT849 plasmids. Screening of mutants was performed in S. lividans and S. rimousus.

Table of Characterized Mutants Mutant Position Substitution Wild-type ebh24-16 92 Valine Isoleucine 237 Alanine Phenylalanine ebh25-1 195 Serine Asparagine 294 Proline Serine ebh10-53 190 Tyrosine Phenylalanine 231 Arginine Glutamic acid ebh24-16d8 92 Valine Isoleucine 237 Alanine Phenylalanine 67 Glutamine Arginine ebh24-16c11 92 Valine Isoleucine 93 Glycine Alanine 237 Alanine Phenylalanine 365 Threonine Isoleucine ebh24-16-16 92 Valine Isoleucine 106 Alanine Valine 237 Alanine Phenylalanine ebh24-16-74 88 Histidine Arginine 92 Valine Isoleucine 237 Alanine Phenylalanine ebh-M18 31 Lysine Glutamic acid 176 Valine Methionine ebh24-16g8 92 Valine Isoleucine 237 Alanine Phenylalanine 67 Glutamine Arginine 130 Threonine Isoleucine 176 Alanine Methionine ebh24-16b9 92 Valine Isoleucine 237 Alanine Phenylalanine 67 Glutamine Arginine 140 Threonine Alanine 176 Serine Methionine

Example 18 Comparison of Epothilone B Transformation in Cells Expressing ebh and Mutants Thereof

In these experiments, twenty ml of YEME medium in a 125 ml bi-indented flask was inoculated with 200 μl of a frozen spore preparation of S. lividans TK24, S. lividans (pANT849-ebh), S. lividans (pANT849-ebh10-53) or S. lividans (pANT849-ebh24-16) and incubated 48 hours at 230 rpm, 30° C. Thiostrepton, 10 μg/ml was added to media inoculated with S. lividans (pANT849-ebh), S. lividans (pANT849-ebh10-53) and S. lividans (pANT849-ebh24-16). Four ml of culture was transferred to 20 ml of R5 medium in a 125 ml Erlenmeyer flask and incubated 18 hrs at 230 rpm, 30° C. Epothilone B in 100% EtOH was added to each culture to a final concentration of 0.05% weight/volume. Samples were taken at 0, 24, 48 and 72 hours with the exception of the S. lividans (pANT849-ebh24-16) culture, in which the epothilone B had been completely converted to epothilone F at 48 hours. The samples were analyzed by HPLC. Results were calculated as a percentage of the epothilone B at time 0 hours.

Epothilone B:

pANT849- Time (hours) TK24 pANT849-ebh pANT849-ebh10-53 ebh24-16 0 100% 100% 100% 100% 24  99%  78%  69%  56% 48  87%  19%  39%  0% 72  87%  0%  3% — Epothilone F:

pANT849- Time (hours) TK24 pANT849-ebh pANT849-ebh10-53 ebh24-16 0 0%  0%  0%  0% 24 0%  4%  9% 23% 48 0% 21% 29% 52% 72 0% 14% 41% —

Alternatively, the bioconversion of epothilone B to epothilone F was performed in S. rimosus host cells transformed with expression plasmids containing the ebh gene and its variants or mutants. One-hundred μl of a frozen S. rimosus transformant culture was inoculated to 20 ml CRM media with 10 μg/ml thiostrepton 10 and cultivated 16–24 hr, 30° C., 230–300 rpm. Epothilone B in 100% ethanol was added to each culture to a final concentration of 0.05% weight/volume. The reaction was typically incubated 20–40 hrs at 30° C., 230–300 rpm. The concentration of epothilones B and F was determined by HPLC analysis.

Evaluation of Mutants in S. rimosus

Mutant Epothilone F yield ebh-M18 55% ebh24-16d8 75% ebh24-16c11 75% ebh24-16-16 75% ebh24-16-74 75% ebh24-16b9 80% ebh24-16g8 85%

Example 19 Biotransformation of Compactin to Pravastatin

Twenty ml of R2YE media with 10 μg/ml thiostrepton in a 125 ml flask was inoculated with 200 μl of a frozen spore preparation of S. lividans (pANT849), S. lividans (pANT849-ebh) and incubated 72 hours at 230 rpm, 28° C. Four ml of culture was inoculated to 20 ml of R2YE media and grown 24 hours at 230 rpm, 28° C. One ml of culture was transferred to a 15 ml polypropylene culture tube, 10 μl of compactin (40 mg/ml) was added to each culture and incubated for 24 hours, 28° C., 250 rpm. Five hundred μl of the culture broth was transferred to a fresh 15 ml polypropylene culture tube. Five hundred μl of 50 mM sodium hydroxide was added and vortexed. Three ml of methanol was added and vortexed, the tube was centrifuged 10 minutes at 3000 rpm in a TJ-6 table-top centrifuge. The organic phase was analyzed by HPLC. Compactin and pravastatin values were assessed relative to the control S. lividans (pANT849) culture.

Compactin and Pravastatin as a Percentage of Starting Compactin Concentration:

S. lividans (pANT849) S. lividans (pANT849-ebh) Compactin 36% 11% Pravastatin 11% 53%

Example 20 High Performance Liquid Chromatography Method for Compactin and Pravastatin Detection

The liquid chromatography separation was performed using a Hewlett Packard1090 Series Separation system (Agilent Technologies, Palo Alto, Calif., USA) and a column, 50×46 mm, filled with Spherisorb ODS2, particle size 5 μM (Keystone Scientific, Inc, Bellefonte, Pa., USA). The gradient mobile phase programming was used with a flow rate of 2.0 ml/minute. Eluent A was water, 10 mM ammonium acetate and 0.05% Phosphoric Acid. Eluent B was acetonitrile. The mobile phase was a linear gradient from 20% B to 90% B over 4 minutes.

Example 21 Structure Determination of the Biotransformation Product of Mutant ebh25-1

Analytical HPLC was performed using a Hewlett Packard 1100 Series Liquid Chromatograph with a YMC Packed ODS-AQ column, 4.6 mm i.d.×15 cm 1. A gradient system of water (solvent A) and acetonitrile (solvent B) was used: 20% to 90% B linear gradient, 10 minutes; 90% to 20% linear gradient, 2 minutes. The flow rate was 1 ml/minute and UV detection was at 254 nm.

Preparative HPLC was performed using the following equipment and conditions:

-   Pump: Varian ProStar Solvent Delivery Module (Varian Inc., Palo     Alto, Calif., USA). Detector: Gynkotek UVD340S. -   Column: YMC ODS-A column (30 mmID×100 mm length, 5μ particle size). -   Elution flow rate: 30 ml/minute -   Elution gradient: (solvent A: water; solvent B: acetonitrile), 20%     B, 2 minutes; 20% to 60% B linear gradient, 18 minutes; 60% B, 2     minutes; 60% to 90% B linear gradient, 1 minute; 90% B, 3 minutes;     90% to 20% B linear gradient, 2 minutes. -   Detection: UV, 210 nm.

LC/NMR was performed as follows: 40 μl of sample was injected onto a YMC Packed ODS-AQ column (4.6 mm i.d.×15 cm 1). The column was eluted at 1 ml/minute flow rate with a gradient system of D₂O (solvent A) and acetonitrile-d₃ (solvent B): 30% B, 1 minute; 30% to 80% B linear gradient, 11 minutes. The eluent passed a UV detection cell (monitored at 254 nm) before flowing through a F19/H₁NMR probe (60 μl active volume) in Varian AS-600 NMR spectrometer. The biotransformation product was eluted at around 7.5 minutes and the flow was stopped manually to allow the eluent to remain in the NMR probe for NMR data acquisition.

Isolation and analysis was performed as follows. The butanol/methanol extract (about 10 ml) was evaporated to dryness under nitrogen stream. One ml methanol was added to the residue (38 mg) and insoluble material was removed by centrifugation (13000 rpm, 2 min). 0.1 ml of the supernatant was used for LC/NMR study and the rest of 0.9 ml was subjected to the preparative HPLC (0.2–0.4 ml per injection). Two major peaks were observed and collected: peak A was eluted between 14 and 15 minutes, while peak B was eluted between 16.5 and 17.5 minutes. Analytical HPLC analysis indicated that peak B was the parent compound, epothilone B (Rt 8.5 minutes), and peak A was the biotransformation product (Rt 7.3 minutes). The peak A fractions were pooled and MS analysis data was obtained with the pooled fractions. The pooled fraction was evaporated to a small volume, then was lyophilized to give 3 mg of white solid. NMR and HPLC analysis of the white solid (dissolved in methanol) revealed that the biotransformation product was partially decomposed during the drying process.

APPENDIX 1 Atom No. Residue Atom Name X-coord Y-coord Z-coord 1 ALA9 N −39.918 −4.913 −1.651 2 ALA9 CA −38.454 −5.033 −1.537 3 ALA9 C −37.953 −4.886 −0.099 4 ALA9 O −38.625 −4.31 0.765 5 ALA9 CB −37.809 −3.967 −2.415 6 THR10 N −36.781 −5.447 0.146 7 THR10 CA −36.187 −5.437 1.49 8 THR10 C −34.916 −4.585 1.553 9 THR10 O −34.016 −4.735 0.72 10 THR10 CB −35.871 −6.887 1.846 11 THR10 OG1 −37.075 −7.631 1.717 12 THR10 CG2 −35.355 −7.053 3.271 13 LEU11 N −34.858 −3.699 2.536 14 LEU11 CA −33.669 −2.853 2.745 15 LEU11 C −32.511 −3.649 3.353 16 LEU11 O −32.706 −4.468 4.259 17 LEU11 CB −34.033 −1.707 3.687 18 LEU11 CG −35.079 −0.78 3.078 19 LEU11 CD1 −35.53 0.265 4.091 20 LEU11 CD2 −34.555 −0.111 1.81 21 PRO12 N −31.32 −3.422 2.823 22 PRO12 CA −30.121 −4.119 3.302 23 PRO12 C −29.652 −3.606 4.663 24 PRO12 O −29.656 −2.397 4.918 25 PRO12 CB −29.081 −3.842 2.259 26 PRO12 CG −29.597 −2.771 1.309 27 PRO12 CD −31.031 −2.493 1.729 28 LEU13 N −29.278 −4.522 5.54 29 LEU13 CA −28.676 −4.118 6.819 30 LEU13 C −27.183 −3.88 6.627 31 LEU13 O −26.449 −4.806 6.267 32 LEU13 CB −28.898 −5.196 7.872 33 LEU13 CG −30.374 −5.354 8.217 34 LEU13 CD1 −30.587 −6.516 9.181 35 LEU13 CD2 −30.945 −4.067 8.802 36 ALA14 N −26.72 −2.741 7.112 37 ALA14 CA −25.355 −2.266 6.825 38 ALA14 C −24.244 −2.941 7.634 39 ALA14 O −23.058 −2.719 7.372 40 ALA14 CB −25.311 −0.764 7.075 41 ARG15 N −24.628 −3.792 8.569 42 ARG15 CA −23.664 −4.537 9.379 43 ARG15 C −23.478 −5.983 8.91 44 ARG15 O −22.815 −6.767 9.599 45 ARG15 CB −24.174 −4.519 10.81 46 ARG15 CG −25.655 −4.879 10.84 47 ARG15 CD −26.2 −4.843 12.26 48 ARG15 NE −27.657 −5.039 12.256 49 ARG15 CZ −28.358 −5.301 13.36 50 ARG15 NH1 −29.69 −5.376 13.3 51 ARG15 NH2 −27.735 −5.412 14.536 52 LYS16 N −24.096 −6.351 7.798 53 LYS16 CA −24.016 −7.741 7.335 54 LYS16 C −22.639 −8.128 6.807 55 LYS16 O −21.959 −7.359 6.115 56 LYS16 CB −25.061 −7.977 6.252 57 LYS16 CG −26.466 −7.985 6.839 58 LYS16 CD −26.605 −9.079 7.892 59 LYS16 CE −28.002 −9.092 8.499 60 LYS16 NZ −28.113 −10.128 9.537 61 CYS17 N −22.317 −9.392 7.036 62 CYS17 CA −21.061 −10.004 6.56 63 CYS17 C −20.737 −9.771 5.066 64 CYS17 O −19.662 −9.205 4.833 65 CYS17 CB −21.096 −11.501 6.864 66 CYS17 SG −21.33 −11.937 8.602 67 PRO18 N −21.635 −10.003 4.1 68 PRO18 CA −21.293 −9.756 2.683 69 PRO18 C −21.123 −8.291 2.246 70 PRO18 O −21.013 −8.061 1.036 71 PRO18 CB −22.388 −10.383 1.878 72 PRO18 CG −23.509 −10.812 2.802 73 PRO18 CD −23.002 −10.554 4.207 74 PHE19 N −21.137 −7.33 3.162 75 PHE19 CA −20.792 −5.947 2.834 76 PHE19 C −19.279 −5.777 2.788 77 PHE19 O −18.789 −4.92 2.036 78 PHE19 CB −21.36 −5.007 3.894 79 PHE19 CG −22.8 −4.568 3.654 80 PHE19 CD1 −23.051 −3.27 3.232 81 PHE19 CD2 −23.856 −5.444 3.867 82 PHE19 CE1 −24.355 −2.853 3.003 83 PHE19 CE2 −25.159 −5.03 3.629 84 PHE19 CZ −25.409 −3.735 3.197 85 SER20 N −18.573 −6.687 3.449 86 SER20 CA −17.102 −6.717 3.446 87 SER20 C −16.569 −7.839 4.342 88 SER20 O −16.632 −7.723 5.573 89 SER20 CB −16.557 −5.371 3.929 90 SER20 OG −17.236 −5.019 5.129 91 PRO21 N −15.974 −8.867 3.753 92 PRO21 CA −15.978 −9.134 2.304 93 PRO21 C −17.267 −9.836 1.856 94 PRO21 O −18.026 −10.327 2.702 95 PRO21 CB −14.8 −10.047 2.111 96 PRO21 CG −14.442 −10.669 3.455 97 PRO21 CD −15.306 −9.949 4.481 98 PRO22 N −17.551 −9.859 0.561 99 PRO22 CA −16.897 −9.007 −0.445 100 PRO22 C −17.4 −7.575 −0.296 101 PRO22 O −18.341 −7.371 0.469 102 PRO22 CB −17.32 −9.591 −1.762 103 PRO22 CG −18.478 −10.549 −1.528 104 PRO22 CD −18.669 −10.604 −0.021 105 PRO23 N −16.687 −6.605 −0.842 106 PRO23 CA −17.224 −5.241 −0.897 107 PRO23 C −18.525 −5.21 −1.693 108 PRO23 O −18.524 −5.083 −2.925 109 PRO23 CB −16.159 −4.417 −1.547 110 PRO23 CG −15.004 −5.321 −1.95 111 PRO23 CD −15.388 −6.725 −1.509 112 GLU24 N −19.62 −5.122 −0.956 113 GLU24 CA −20.963 −5.192 −1.547 114 GLU24 C −21.415 −3.843 −2.088 115 GLU24 O −22.323 −3.794 −2.93 116 GLU24 CB −21.934 −5.68 −0.48 117 GLU24 CG −23.27 −6.137 −1.052 118 GLU24 CD −23.982 −7.017 −0.024 119 GLU24 OE1 −24.613 −7.981 −0.433 120 GLU24 OE2 −23.833 −6.745 1.158 121 TYR25 N −20.573 −2.843 −1.878 122 TYR25 CA −20.842 −1.47 −2.303 123 TYR25 C −20.704 −1.311 −3.816 124 TYR25 O −21.364 −0.436 −4.385 125 TYR25 CB −19.828 −0.568 −1.608 126 TYR25 CG −19.616 −0.882 −0.128 127 TYR25 CD1 −20.662 −0.753 0.779 128 TYR25 CD2 −18.364 −1.298 0.311 129 TYR25 CE1 −20.461 −1.062 2.119 130 TYR25 CE2 −18.163 −1.605 1.65 131 TYR25 CZ −19.213 −1.492 2.55 132 TYR25 OH −19.026 −1.859 3.866 133 GLU26 N −20.1 −2.296 −4.468 134 GLU26 CA −20.009 −2.293 −5.928 135 GLU26 C −21.404 −2.483 −6.52 136 GLU26 O −21.92 −1.572 −7.177 137 GLU26 CB −19.129 −3.454 −6.39 138 GLU26 CG −17.813 −3.593 −5.628 139 GLU26 CD −16.94 −2.342 −5.707 140 GLU26 OE1 −16.345 −2.12 −6.749 141 GLU26 OE2 −16.773 −1.731 −4.657 142 ARG27 N −22.105 −3.488 −6.017 143 ARG27 CA −23.437 −3.805 −6.538 144 ARG27 C −24.504 −2.909 −5.921 145 ARG27 O −25.496 −2.591 −6.59 146 ARG27 CB −23.752 −5.26 −6.22 147 ARG27 CG −22.7 −6.189 −6.812 148 ARG27 CD −23.031 −7.653 −6.55 149 ARG27 NE −23.146 −7.926 −5.108 150 ARG27 CZ −22.251 −8.648 −4.428 151 ARG27 NH1 −21.16 −9.11 −5.043 152 ARG27 NH2 −22.428 −8.879 −3.126 153 LEU28 N −24.197 −2.331 −4.771 154 LEU28 CA −25.11 −1.358 −4.168 155 LEU28 C −25.131 −0.079 −4.987 156 LEU28 O −26.214 0.286 −5.45 157 LEU28 CB −24.67 −1.039 −2.746 158 LEU28 CG −24.868 −2.224 −1.81 159 LEU28 CD1 −24.303 −1.916 −0.43 160 LEU28 CD2 −26.34 −2.609 −1.716 161 ARG29 N −23.969 0.307 −5.49 162 ARG29 CA −23.835 1.502 −6.327 163 ARG29 C −24.521 1.334 −7.677 164 ARG29 O −25.271 2.226 −8.096 165 ARG29 CB −22.345 1.682 −6.568 166 ARG29 CG −21.997 2.947 −7.336 167 ARG29 CD −20.519 2.941 −7.711 168 ARG29 NE −19.696 2.563 −6.551 169 ARG29 CZ −18.945 1.459 −6.523 170 ARG29 NH1 −18.872 0.673 −7.6 171 ARG29 NH2 −18.265 1.145 −5.421 172 ARG30 N −24.494 0.109 −8.182 173 ARG30 CA −25.112 −0.208 −9.475 174 ARG30 C −26.629 −0.386 −9.407 175 ARG30 O −27.282 −0.429 −10.455 176 ARG30 CB −24.503 −1.512 −9.971 177 ARG30 CG −22.992 −1.401 −10.1 178 ARG30 CD −22.376 −2.745 −10.463 179 ARG30 NE −20.909 −2.659 −10.479 180 ARG30 CZ −20.12 −3.648 −10.054 181 ARG30 NH1 −20.658 −4.772 −9.576 182 ARG30 NH2 −18.793 −3.508 −10.099 183 GLU31 N −27.194 −0.493 −8.215 184 GLU31 CA −28.653 −0.576 −8.109 185 GLU31 C −29.207 0.713 −7.51 186 GLU31 O −30.393 1.032 −7.656 187 GLU31 CB −29.025 −1.746 −7.203 188 GLU31 CG −28.381 −3.055 −7.65 189 GLU31 CD −28.814 −3.443 −9.061 190 GLU31 OE1 −30.013 −3.448 −9.301 191 GLU31 OE2 −27.961 −3.944 −9.782 192 SER32 N −28.319 1.439 −6.855 193 SER32 CA −28.652 2.672 −6.147 194 SER32 C −27.386 3.393 −5.683 195 SER32 O −26.706 2.984 −4.731 196 SER32 CB −29.509 2.309 −4.939 197 SER32 OG −28.842 1.268 −4.234 198 PRO33 N −27.148 4.543 −6.292 199 PRO33 CA −26.039 5.408 −5.869 200 PRO33 C −26.227 5.972 −4.454 201 PRO33 O −25.241 6.254 −3.758 202 PRO33 CB −26.023 6.511 −6.879 203 PRO33 CG −27.203 6.364 −7.829 204 PRO33 CD −27.933 5.107 −7.394 205 VAL34 N −27.478 6.094 −4.033 206 VAL34 CA −27.83 6.472 −2.661 207 VAL34 C −28.828 5.447 −2.122 208 VAL34 O −30.01 5.467 −2.487 209 VAL34 CB −28.483 7.85 −2.686 210 VAL34 CG1 −28.789 8.339 −1.275 211 VAL34 CG2 −27.616 8.865 −3.42 212 SER35 N −28.344 4.546 −1.286 213 SER35 CA −29.186 3.438 −0.802 214 SER35 C −29.512 3.536 0.688 215 SER35 O −28.615 3.692 1.521 216 SER35 CB −28.456 2.126 −1.077 217 SER35 OG −27.19 2.169 −0.43 218 ARG36 N −30.785 3.413 1.025 219 ARG36 CA −31.168 3.431 2.443 220 ARG36 C −30.894 2.072 3.082 221 ARG36 O −31.516 1.059 2.741 222 ARG36 CB −32.645 3.779 2.597 223 ARG36 CG −33.016 3.857 4.076 224 ARG36 CD −34.513 4.047 4.295 225 ARG36 NE −34.987 5.35 3.804 226 ARG36 CZ −36.272 5.582 3.523 227 ARG36 NH1 −37.16 4.59 3.609 228 ARG36 NH2 −36.662 6.791 3.113 229 VAL37 N −29.921 2.067 3.974 230 VAL37 CA −29.543 0.855 4.695 231 VAL37 C −29.982 0.926 6.152 232 VAL37 O −30.313 1.995 6.684 233 VAL37 CB −28.03 0.681 4.608 234 VAL37 CG1 −27.591 0.391 3.177 235 VAL37 CG2 −27.298 1.898 5.163 236 GLY38 N −30.064 −0.24 6.761 237 GLY38 CA −30.404 −0.332 8.18 238 GLY38 C −29.151 −0.563 9.016 239 GLY38 O −28.562 −1.652 9.003 240 LEU39 N −28.764 0.463 9.75 241 LEU39 CA −27.607 0.399 10.656 242 LEU39 C −27.911 −0.554 11.817 243 LEU39 O −29.028 −1.085 11.882 244 LEU39 CB −27.353 1.814 11.187 245 LEU39 CG −26.198 2.546 10.5 246 LEU39 CD1 −26.368 2.665 8.988 247 LEU39 CD2 −26.011 3.925 11.12 248 PRO40 N −26.919 −0.869 12.643 249 PRO40 CA −27.183 −1.62 13.875 250 PRO40 C −28.423 −1.116 14.614 251 PRO40 O −28.771 0.073 14.574 252 PRO40 CB −25.933 −1.51 14.691 253 PRO40 CG −24.84 −0.886 13.837 254 PRO40 CD −25.497 −0.52 12.516 255 SER41 N −29.188 −2.109 15.042 256 SER41 CA −30.511 −1.986 15.686 257 SER41 C −31.548 −1.213 14.856 258 SER41 O −32.379 −0.492 15.419 259 SER41 CB −30.387 −1.382 17.087 260 SER41 OG −30.036 −0.008 17.001 261 GLY42 N −31.474 −1.34 13.539 262 GLY42 CA −32.521 −0.831 12.644 263 GLY42 C −32.557 0.686 12.434 264 GLY42 O −33.59 1.208 11.997 265 GLN43 N −31.471 1.392 12.713 266 GLN43 CA −31.501 2.847 12.494 267 GLN43 C −31.201 3.16 11.025 268 GLN43 O −30.079 2.955 10.551 269 GLN43 CB −30.507 3.53 13.437 270 GLN43 CG −30.681 5.05 13.439 271 GLN43 CD −29.873 5.699 14.567 272 GLN43 OE1 −30.31 6.682 15.184 273 GLN43 NE2 −28.723 5.116 14.852 274 THR44 N −32.227 3.582 10.304 275 THR44 CA −32.096 3.832 8.859 276 THR44 C −31.194 5.02 8.534 277 THR44 O −31.231 6.071 9.187 278 THR44 CB −33.475 4.077 8.258 279 THR44 OG1 −34.009 5.268 8.823 280 THR44 CG2 −34.428 2.923 8.551 281 ALA45 N −30.35 4.799 7.541 282 ALA45 CA −29.426 5.833 7.07 283 ALA45 C −29.16 5.718 5.572 284 ALA45 O −29.105 4.619 5.009 285 ALA45 CB −28.115 5.705 7.836 286 TRP46 N −28.989 6.859 4.931 287 TRP46 CA −28.702 6.865 3.492 288 TRP46 C −27.212 6.698 3.221 289 TRP46 O −26.408 7.589 3.517 290 TRP46 CB −29.185 8.173 2.881 291 TRP46 CG −30.693 8.309 2.805 292 TRP46 CD1 −31.509 9.009 3.665 293 TRP46 CD2 −31.552 7.723 1.804 294 TRP46 NE1 −32.788 8.894 3.228 295 TRP46 CE2 −32.862 8.146 2.116 296 TRP46 CE3 −31.324 6.922 0.701 297 TRP46 CZ2 −33.913 7.774 1.295 298 TRP46 CZ3 −32.389 6.538 −0.105 299 TRP46 CH2 −33.68 6.967 0.19 300 ALA47 N −26.863 5.559 2.652 301 ALA47 CA −25.475 5.257 2.302 302 ALA47 C −25.153 5.708 0.882 303 ALA47 O −25.772 5.272 −0.1 304 ALA47 CB −25.248 3.756 2.427 305 LEU48 N −24.185 6.602 0.797 306 LEU48 CA −23.751 7.129 −0.501 307 LEU48 C −22.648 6.252 −1.067 308 LEU48 O −21.546 6.197 −0.511 309 LEU48 CB −23.222 8.543 −0.317 310 LEU48 CG −24.27 9.464 0.289 311 LEU48 CD1 −23.707 10.863 0.454 312 LEU48 CD2 −25.524 9.515 −0.569 313 THR49 N −22.948 5.601 −2.176 314 THR49 CA −22.01 4.636 −2.75 315 THR49 C −21.197 5.214 −3.907 316 THR49 O −20.047 4.803 −4.09 317 THR49 CB −22.774 3.391 −3.196 318 THR49 OG1 −23.783 3.769 −4.125 319 THR49 CG2 −23.458 2.703 −2.02 320 ARG50 N −21.724 6.2 −4.616 321 ARG50 CA −20.899 6.838 −5.655 322 ARG50 C −20.007 7.927 −5.081 323 ARG50 O −20.456 8.712 −4.234 324 ARG50 CB −21.737 7.467 −6.758 325 ARG50 CG −22.426 6.441 −7.639 326 ARG50 CD −22.852 7.085 −8.951 327 ARG50 NE −23.597 8.327 −8.704 328 ARG50 CZ −23.779 9.27 −9.629 329 ARG50 NH1 −24.462 10.375 −9.326 330 ARG50 NH2 −23.274 9.111 −10.854 331 LEU51 N −18.92 8.175 −5.797 332 LEU51 CA −17.931 9.19 −5.399 333 LEU51 C −18.52 10.584 −5.583 334 LEU51 O −18.42 11.426 −4.682 335 LEU51 CB −16.726 9.066 −6.33 336 LEU51 CG −15.377 9.193 −5.621 337 LEU51 CD1 −14.233 9.154 −6.628 338 LEU51 CD2 −15.267 10.433 −4.746 339 GLU52 N −19.404 10.68 −6.562 340 GLU52 CA −20.088 11.93 −6.891 341 GLU52 C −21.101 12.314 −5.811 342 GLU52 O −21.114 13.477 −5.389 343 GLU52 CB −20.821 11.759 −8.229 344 GLU52 CG −19.897 11.56 −9.439 345 GLU52 CD −19.749 10.09 −9.853 346 GLU52 OE1 −19.796 9.24 −8.971 347 GLU52 OE2 −19.502 9.849 −11.025 348 ASP53 N −21.659 11.313 −5.146 349 ASP53 CA −22.646 11.572 −4.096 350 ASP53 C −21.953 11.905 −2.783 351 ASP53 O −22.4 12.804 −2.063 352 ASP53 CB −23.493 10.322 −3.876 353 ASP53 CG −24.263 9.94 −5.133 354 ASP53 OD1 −24.319 8.749 −5.405 355 ASP53 OD2 −24.633 10.838 −5.878 356 ILE54 N −20.75 11.382 −2.614 357 ILE54 CA −19.991 11.62 −1.387 358 ILE54 C −19.301 12.976 −1.41 359 ILE54 O −19.36 13.7 −0.409 360 ILE54 CB −18.963 10.509 −1.269 361 ILE54 CG1 −19.674 9.167 −1.252 362 ILE54 CG2 −18.113 10.671 −0.015 363 ILE54 CD1 −18.677 8.03 −1.365 364 ARG55 N −18.916 13.43 −2.592 365 ARG55 CA −18.346 14.776 −2.704 366 ARG55 C −19.44 15.836 −2.679 367 ARG55 O −19.252 16.893 −2.065 368 ARG55 CB −17.551 14.883 −3.998 369 ARG55 CG −16.293 14.028 −3.94 370 ARG55 CD −15.498 14.133 −5.235 371 ARG55 NE −16.277 13.61 −6.367 372 ARG55 CZ −15.712 13.028 −7.427 373 ARG55 NH1 −14.383 12.947 −7.513 374 ARG55 NH2 −16.475 12.553 −8.413 375 GLU56 N −20.64 15.438 −3.068 376 GLU56 CA −21.795 16.331 −2.984 377 GLU56 C −22.287 16.444 −1.539 378 GLU56 O −22.628 17.546 −1.095 379 GLU56 CB −22.875 15.722 −3.866 380 GLU56 CG −24.103 16.605 −4.028 381 GLU56 CD −25.112 15.838 −4.874 382 GLU56 OE1 −25.906 16.463 −5.56 383 GLU56 OE2 −25.055 14.616 −4.834 384 MET57 N −22.065 15.392 −0.767 385 MET57 CA −22.379 15.386 0.665 386 MET57 C −21.4 16.241 1.459 387 MET57 O −21.827 17.091 2.248 388 MET57 CB −22.242 13.948 1.141 389 MET57 CG −22.423 13.805 2.646 390 MET57 SD −21.979 12.184 3.306 391 MET57 CE −20.221 12.196 2.89 392 LEU58 N −20.14 16.197 1.056 393 LEU58 CA −19.089 16.973 1.726 394 LEU58 C −19.07 18.444 1.307 395 LEU58 O −18.398 19.263 1.946 396 LEU58 CB −17.751 16.327 1.389 397 LEU58 CG −17.638 14.941 2.013 398 LEU58 CD1 −16.504 14.133 1.394 399 LEU58 CD2 −17.49 15.03 3.528 400 SER59 N −19.807 18.776 0.261 401 SER59 CA −19.959 20.171 −0.144 402 SER59 C −21.305 20.739 0.304 403 SER59 O −21.531 21.951 0.204 404 SER59 CB −19.852 20.24 −1.661 405 SER59 OG −18.59 19.697 −2.022 406 SER60 N −22.175 19.879 0.807 407 SER60 CA −23.5 20.318 1.246 408 SER60 C −23.505 20.806 2.685 409 SER60 O −23.464 19.996 3.62 410 SER60 CB −24.477 19.156 1.138 411 SER60 OG −25.689 19.581 1.749 412 PRO61 N −23.91 22.058 2.835 413 PRO61 CA −24.023 22.695 4.154 414 PRO61 C −25.231 22.233 4.983 415 PRO61 O −25.41 22.7 6.113 416 PRO61 CB −24.145 24.157 3.853 417 PRO61 CG −24.401 24.343 2.364 418 PRO61 CD −24.301 22.959 1.747 419 HIS62 N −26.044 21.333 4.451 420 HIS62 CA −27.21 20.856 5.18 421 HIS62 C −26.949 19.497 5.813 422 HIS62 O −27.863 18.935 6.427 423 HIS62 CB −28.379 20.764 4.214 424 HIS62 CG −28.703 22.084 3.55 425 HIS62 ND1 −28.955 23.252 4.171 426 HIS62 CD2 −28.796 22.32 2.198 427 HIS62 CE1 −29.197 24.205 3.248 428 HIS62 NE2 −29.098 23.627 2.029 429 PHE63 N −25.765 18.945 5.596 430 PHE63 CA −25.385 17.693 6.258 431 PHE63 C −24.492 17.977 7.456 432 PHE63 O −23.261 17.885 7.396 433 PHE63 CB −24.686 16.783 5.262 434 PHE63 CG −25.651 16.13 4.284 435 PHE63 CD1 −26.92 15.76 4.71 436 PHE63 CD2 −25.265 15.901 2.972 437 PHE63 CE1 −27.804 15.161 3.824 438 PHE63 CE2 −26.147 15.298 2.087 439 PHE63 CZ −27.415 14.928 2.512 440 SER64 N −25.159 18.211 8.569 441 SER64 CA −24.502 18.656 9.795 442 SER64 C −23.765 17.525 10.507 443 SER64 O −24.07 16.34 10.339 444 SER64 CB −25.587 19.225 10.7 445 SER64 OG −24.96 19.785 11.84 446 SER65 N −22.719 17.898 11.218 447 SER65 CA −22.006 16.938 12.053 448 SER65 C −22.463 17.032 13.513 449 SER65 O −22.031 16.247 14.365 450 SER65 CB −20.522 17.234 11.936 451 SER65 OG −20.167 17.174 10.564 452 ASP66 N −23.368 17.961 13.782 453 ASP66 CA −23.901 18.186 15.122 454 ASP66 C −25.388 18.496 14.919 455 ASP66 O −25.978 18.026 13.938 456 ASP66 CB −23.149 19.393 15.69 457 ASP66 CG −22.904 19.311 17.192 458 ASP66 OD1 −21.835 19.724 17.618 459 ASP66 OD2 −23.871 19.048 17.899 460 ARG67 N −25.972 19.246 15.842 461 ARG67 CA −27.32 19.831 15.692 462 ARG67 C −28.423 18.78 15.619 463 ARG67 O −28.768 18.296 14.533 464 ARG67 CB −27.384 20.684 14.423 465 ARG67 CG −26.263 21.716 14.336 466 ARG67 CD −26.329 22.778 15.428 467 ARG67 NE −25.137 23.64 15.358 468 ARG67 CZ −25.091 24.799 14.695 469 ARG67 NH1 −26.189 25.28 14.107 470 ARG67 NH2 −23.957 25.503 14.663 471 GLN68 N −28.983 18.45 16.768 472 GLN68 CA −30.127 17.538 16.79 473 GLN68 C −31.414 18.348 16.65 474 GLN68 O −31.728 19.187 17.503 475 GLN68 CB −30.116 16.757 18.1 476 GLN68 CG −31.207 15.692 18.12 477 GLN68 CD −31.109 14.852 19.389 478 GLN68 OE1 −31.941 14.973 20.296 479 GLN68 NE2 −30.137 13.955 19.406 480 SER69 N −32.129 18.102 15.565 481 SER69 CA −33.37 18.833 15.272 482 SER69 C −34.444 18.558 16.32 483 SER69 O −34.447 17.495 16.958 484 SER69 CB −33.885 18.387 13.91 485 SER69 OG −34.261 17.025 14.033 486 PRO70 N −35.332 19.526 16.499 487 PRO70 CA −36.438 19.385 17.447 488 PRO70 C −37.244 18.122 17.171 489 PRO70 O −37.547 17.795 16.018 490 PRO70 CB −37.267 20.622 17.291 491 PRO70 CG −36.6 21.547 16.285 492 PRO70 CD −35.348 20.824 15.815 493 SER71 N −37.424 17.369 18.245 494 SER71 CA −38.115 16.065 18.289 495 SER71 C −37.589 15.02 17.298 496 SER71 O −38.378 14.228 16.769 497 SER71 CB −39.625 16.244 18.111 498 SER71 OG −39.919 16.638 16.777 499 PHE72 N −36.282 14.985 17.081 500 PHE72 CA −35.679 13.876 16.321 501 PHE72 C −34.364 13.43 16.957 502 PHE72 O −33.281 13.768 16.458 503 PHE72 CB −35.428 14.283 14.872 504 PHE72 CG −36.682 14.456 14.018 505 PHE72 CD1 −37.097 15.724 13.63 506 PHE72 CD2 −37.402 13.339 13.617 507 PHE72 CE1 −38.238 15.875 12.853 508 PHE72 CE2 −38.544 13.489 12.84 509 PHE72 CZ −38.962 14.758 12.459 510 PRO73 N −34.469 12.59 17.979 511 PRO73 CA −33.31 12.19 18.786 512 PRO73 C −32.522 11.027 18.18 513 PRO73 O −32.606 9.895 18.668 514 PRO73 CB −33.898 11.776 20.099 515 PRO73 CG −35.392 11.555 19.917 516 PRO73 CD −35.708 12.004 18.5 517 LEU74 N −31.772 11.304 17.127 518 LEU74 CA −30.933 10.263 16.521 519 LEU74 C −29.707 9.976 17.375 520 LEU74 O −29.08 10.892 17.926 521 LEU74 CB −30.474 10.697 15.135 522 LEU74 CG −31.627 10.794 14.146 523 LEU74 CD1 −31.094 11.194 12.776 524 LEU74 CD2 −32.381 9.471 14.05 525 MET75 N −29.359 8.705 17.454 526 MET75 CA −28.167 8.306 18.208 527 MET75 C −27.099 7.808 17.243 528 MET75 O −27.166 6.675 16.746 529 MET75 CB −28.539 7.208 19.198 530 MET75 CG −27.367 6.867 20.114 531 MET75 SD −27.678 5.549 21.31 532 MET75 CE −28.002 4.197 20.154 533 VAL76 N −26.117 8.657 16.992 534 VAL76 CA −25.071 8.327 16.017 535 VAL76 C −24.274 7.103 16.455 536 VAL76 O −23.953 6.925 17.636 537 VAL76 CB −24.151 9.527 15.809 538 VAL76 CG1 −24.904 10.676 15.149 539 VAL76 CG2 −23.504 9.986 17.109 540 ALA77 N −23.836 6.34 15.467 541 ALA77 CA −23.158 5.062 15.727 542 ALA77 C −21.703 5.203 16.177 543 ALA77 O −21.033 4.194 16.42 544 ALA77 CB −23.22 4.212 14.465 545 ARG78 N −21.218 6.431 16.271 546 ARG78 CA −19.868 6.689 16.762 547 ARG78 C −19.868 7.178 18.215 548 ARG78 O −18.816 7.163 18.865 549 ARG78 CB −19.274 7.772 15.874 550 ARG78 CG −19.445 7.436 14.398 551 ARG78 CD −19.068 8.629 13.528 552 ARG78 NE −19.848 9.81 13.932 553 ARG78 CZ −19.36 11.053 13.921 554 ARG78 NH1 −18.114 11.278 13.497 555 ARG78 NH2 −20.12 12.072 14.33 556 GLN79 N −21.028 7.577 18.722 557 GLN79 CA −21.128 8.129 20.088 558 GLN79 C −22.484 7.818 20.715 559 GLN79 O −23.48 8.503 20.45 560 GLN79 CB −20.937 9.651 20.09 561 GLN79 CG −19.486 10.085 19.884 562 GLN79 CD −19.353 11.607 19.931 563 GLN79 OE1 −19.071 12.193 20.986 564 GLN79 NE2 −19.508 12.226 18.773 565 ILE80 N −22.504 6.806 21.562 566 ILE80 CA −23.733 6.44 22.273 567 ILE80 C −23.732 7.034 23.679 568 ILE80 O −22.666 7.316 24.24 569 ILE80 CB −23.847 4.919 22.333 570 ILE80 CG1 −22.684 4.305 23.109 571 ILE80 CG2 −23.905 4.35 20.92 572 ILE80 CD1 −22.794 2.788 23.191 573 ARG81 N −24.932 7.278 24.188 574 ARG81 CA −25.15 7.84 25.535 575 ARG81 C −24.657 9.276 25.691 576 ARG81 O −23.493 9.571 25.411 577 ARG81 CB −24.51 6.964 26.603 578 ARG81 CG −25.437 5.843 27.046 579 ARG81 CD −25.685 5.92 28.555 580 ARG81 NE −26.269 7.22 28.93 581 ARG81 CZ −25.651 8.095 29.722 582 ARG81 NH1 −24.439 7.82 30.204 583 ARG81 NH2 −26.234 9.257 30.008 584 ARG82 N −25.448 10.076 26.389 585 ARG82 CA −25.192 11.523 26.511 586 ARG82 C −23.872 11.866 27.204 587 ARG82 O −23.108 12.684 26.682 588 ARG82 CB −26.32 12.122 27.333 589 ARG82 CG −27.683 11.796 26.74 590 ARG82 CD −28.801 12.301 27.643 591 ARG82 NE −28.71 11.659 28.967 592 ARG82 CZ −28.623 12.34 30.114 593 ARG82 NH1 −28.477 11.689 31.271 594 ARG82 NH2 −28.606 13.675 30.096 595 GLU83 N −23.495 11.077 28.198 596 GLU83 CA −22.237 11.334 28.909 597 GLU83 C −21.03 10.69 28.227 598 GLU83 O −19.894 10.902 28.657 599 GLU83 CB −22.361 10.828 30.338 600 GLU83 CG −23.385 11.651 31.114 601 GLU83 CD −23.478 11.172 32.56 602 GLU83 OE1 −23.428 9.967 32.761 603 GLU83 OE2 −23.71 12.011 33.418 604 ASP84 N −21.274 9.941 27.165 605 ASP84 CA −20.201 9.327 26.386 606 ASP84 C −20.095 10.012 25.024 607 ASP84 O −19.257 9.646 24.19 608 ASP84 CB −20.481 7.841 26.237 609 ASP84 CG −20.585 7.191 27.613 610 ASP84 OD1 −19.547 6.906 28.193 611 ASP84 OD2 −21.704 7.048 28.092 612 LYS85 N −20.939 11.017 24.831 613 LYS85 CA −20.846 11.928 23.681 614 LYS85 C −19.997 13.228 23.804 615 LYS85 O −20.236 14.084 22.942 616 LYS85 CB −22.27 12.347 23.327 617 LYS85 CG −23.107 11.173 22.832 618 LYS85 CD −24.573 11.567 22.679 619 LYS85 CE −25.408 10.408 22.148 620 LYS85 NZ −26.824 10.785 22.036 621 PRO86 N −19.054 13.455 24.73 622 PRO86 CA −18.316 14.731 24.698 623 PRO86 C −17.168 14.8 23.679 624 PRO86 O −16.339 15.713 23.772 625 PRO86 CB −17.779 14.922 26.08 626 PRO86 CG −17.866 13.6 26.815 627 PRO86 CD −18.543 12.646 25.852 628 PHE87 N −17.14 13.906 22.701 629 PHE87 CA −16.12 13.963 21.653 630 PHE87 C −16.67 14.817 20.51 631 PHE87 O −15.963 15.16 19.559 632 PHE87 CB −15.848 12.556 21.128 633 PHE87 CG −15.724 11.449 22.174 634 PHE87 CD1 −16.447 10.277 21.996 635 PHE87 CD2 −14.904 11.591 23.286 636 PHE87 CE1 −16.358 9.254 22.93 637 PHE87 CE2 −14.817 10.567 24.22 638 PHE87 CZ −15.544 9.399 24.044 639 ARG88 N −17.948 15.143 20.627 640 ARG88 CA −18.629 16.037 19.686 641 ARG88 C −18.178 17.519 19.7 642 ARG88 O −18.118 18.064 18.59 643 ARG88 CB −20.122 15.915 19.965 644 ARG88 CG −20.964 16.678 18.953 645 ARG88 CD −22.429 16.294 19.089 646 ARG88 NE −22.593 14.851 18.868 647 ARG88 CZ −23.307 14.07 19.679 648 ARG88 NH1 −23.373 12.757 19.45 649 ARG88 NH2 −23.922 14.598 20.739 650 PRO89 N −17.919 18.194 20.826 651 PRO89 CA −17.186 19.48 20.763 652 PRO89 C −15.737 19.333 20.277 653 PRO89 O −14.786 19.454 21.057 654 PRO89 CB −17.206 20.033 22.154 655 PRO89 CG −17.798 19.004 23.096 656 PRO89 CD −18.208 17.832 22.224 657 SER90 N −15.606 19.29 18.963 658 SER90 CA −14.334 19.04 18.296 659 SER90 C −14.43 19.413 16.824 660 SER90 O −15.534 19.592 16.293 661 SER90 CB −14.069 17.55 18.375 662 SER90 OG −15.095 16.928 17.609 663 LEU91 N −13.326 19.173 16.14 664 LEU91 CA −13.154 19.564 14.737 665 LEU91 C −14.042 18.804 13.745 666 LEU91 O −14.491 19.39 12.754 667 LEU91 CB −11.702 19.238 14.405 668 LEU91 CG −11.325 19.637 12.988 669 LEU91 CD1 −11.253 21.153 12.877 670 LEU91 CD2 −9.989 19.012 12.606 671 ILE92 N −14.422 17.582 14.076 672 ILE92 CA −15.199 16.791 13.12 673 ILE92 C −16.712 16.857 13.368 674 ILE92 O −17.487 16.497 12.474 675 ILE92 CB −14.682 15.351 13.173 676 ILE92 CG1 −15.288 14.488 12.07 677 ILE92 CG2 −14.932 14.719 14.539 678 ILE92 CD1 −14.775 13.055 12.137 679 ALA93 N −17.14 17.407 14.494 680 ALA93 CA −18.578 17.414 14.759 681 ALA93 C −19.15 18.822 14.872 682 ALA93 O −20.335 19.048 14.589 683 ALA93 CB −18.865 16.593 16.004 684 MET94 N −18.294 19.776 15.191 685 MET94 CA −18.739 21.168 15.216 686 MET94 C −18.99 21.683 13.811 687 MET94 O −18.221 21.436 12.88 688 MET94 CB −17.695 22.042 15.893 689 MET94 CG −17.822 21.982 17.407 690 MET94 SD −16.686 23.058 18.31 691 MET94 CE −17.561 23.095 19.891 692 ASP95 N −20.089 22.398 13.672 693 ASP95 CA −20.42 23.024 12.393 694 ASP95 C −19.831 24.427 12.371 695 ASP95 O −19.589 25.001 13.438 696 ASP95 CB −21.938 23.082 12.258 697 ASP95 CG −22.52 21.677 12.373 698 ASP95 OD1 −22.276 20.87 11.484 699 ASP95 OD2 −23.173 21.412 13.37 700 PRO96 N −19.488 24.936 11.201 701 PRO96 CA −19.076 26.343 11.099 702 PRO96 C −20.177 27.263 11.641 703 PRO96 O −21.353 26.892 11.581 704 PRO96 CB −18.812 26.567 9.64 705 PRO96 CG −19.078 25.278 8.875 706 PRO96 CD −19.532 24.256 9.905 707 PRO97 N −19.817 28.38 12.263 708 PRO97 CA −18.428 28.85 12.436 709 PRO97 C −17.649 28.271 13.631 710 PRO97 O −16.462 28.595 13.772 711 PRO97 CB −18.567 30.329 12.615 712 PRO97 CG −20.013 30.649 12.964 713 PRO97 CD −20.777 29.345 12.804 714 GLU98 N −18.233 27.353 14.389 715 GLU98 CA −17.555 26.761 15.556 716 GLU98 C −16.353 25.946 15.099 717 GLU98 O −15.224 26.174 15.555 718 GLU98 CB −18.513 25.782 16.223 719 GLU98 CG −19.894 26.372 16.474 720 GLU98 CD −20.857 25.236 16.811 721 GLU98 OE1 −20.697 24.171 16.227 722 GLU98 OE2 −21.799 25.49 17.544 723 HIS99 N −16.575 25.223 14.013 724 HIS99 CA −15.52 24.448 13.359 725 HIS99 C −14.43 25.323 12.743 726 HIS99 O −13.249 24.993 12.887 727 HIS99 CB −16.19 23.646 12.252 728 HIS99 CG −15.238 23.09 11.22 729 HIS99 ND1 −14.522 21.957 11.317 730 HIS99 CD2 −14.946 23.649 9.998 731 HIS99 CE1 −13.779 21.805 10.203 732 HIS99 NE2 −14.042 22.852 9.39 733 GLY100 N −14.792 26.524 12.322 734 GLY100 CA −13.832 27.439 11.702 735 GLY100 C −12.859 27.944 12.756 736 GLY100 O −11.648 27.716 12.64 737 LYS101 N −13.419 28.385 13.872 738 LYS101 CA −12.626 28.895 14.993 739 LYS101 C −11.711 27.823 15.579 740 LYS101 O −10.485 28.02 15.622 741 LYS101 CB −13.608 29.33 16.07 742 LYS101 CG −12.893 29.892 17.291 743 LYS101 CD −13.829 29.939 18.492 744 LYS101 CE −14.189 28.531 18.955 745 LYS101 NZ −12.986 27.796 19.381 746 ALA102 N −12.251 26.624 15.738 747 ALA102 CA −11.474 25.523 16.313 748 ALA102 C −10.381 25.022 15.373 749 ALA102 O −9.243 24.859 15.828 750 ALA102 CB −12.425 24.379 16.645 751 ARG103 N −10.622 25.091 14.074 752 ARG103 CA −9.63 24.64 13.097 753 ARG103 C −8.492 25.644 12.958 754 ARG103 O −7.325 25.236 13.033 755 ARG103 CB −10.347 24.476 11.762 756 ARG103 CG −9.496 23.785 10.705 757 ARG103 CD −10.366 23.455 9.496 758 ARG103 NE −9.651 22.682 8.467 759 ARG103 CZ −9.807 21.367 8.287 760 ARG103 NH1 −10.493 20.645 9.175 761 ARG103 NH2 −9.174 20.755 7.285 762 ARG104 N −8.811 26.923 13.087 763 ARG104 CA −7.775 27.957 13.006 764 ARG104 C −6.906 27.966 14.256 765 ARG104 O −5.675 28.083 14.149 766 ARG104 CB −8.442 29.316 12.84 767 ARG104 CG −9.166 29.412 11.502 768 ARG104 CD −9.828 30.772 11.319 769 ARG104 NE −10.874 30.999 12.329 770 ARG104 CZ −11.061 32.171 12.941 771 ARG104 NH1 −10.231 33.188 12.701 772 ARG104 NH2 −12.048 32.31 13.829 773 ASP105 N −7.5 27.625 15.388 774 ASP105 CA −6.718 27.493 16.616 775 ASP105 C −5.828 26.253 16.594 776 ASP105 O −4.602 26.392 16.716 777 ASP105 CB −7.67 27.427 17.806 778 ASP105 CG −8.198 28.814 18.165 779 ASP105 OD1 −7.389 29.588 18.666 780 ASP105 OD2 −9.411 28.938 18.257 781 VAL106 N −6.379 25.125 16.173 782 VAL106 CA −5.636 23.86 16.236 783 VAL106 C −4.51 23.761 15.214 784 VAL106 O −3.414 23.348 15.612 785 VAL106 CB −6.611 22.703 16.046 786 VAL106 CG1 −5.886 21.37 15.89 787 VAL106 CG2 −7.587 22.637 17.212 788 VAL107 N −4.641 24.427 14.075 789 VAL107 CA −3.565 24.397 13.071 790 VAL107 C −2.362 25.259 13.474 791 VAL107 O −1.225 24.918 13.123 792 VAL107 CB −4.142 24.869 11.737 793 VAL107 CG1 −3.06 25.104 10.687 794 VAL107 CG2 −5.175 23.879 11.213 795 GLY108 N −2.576 26.155 14.426 796 GLY108 CA −1.49 26.985 14.953 797 GLY108 C −0.511 26.183 15.813 798 GLY108 O 0.685 26.491 15.837 799 GLU109 N −1.006 25.191 16.537 800 GLU109 CA −0.109 24.388 17.376 801 GLU109 C 0.121 22.976 16.836 802 GLU109 O 1.086 22.311 17.229 803 GLU109 CB −0.677 24.35 18.784 804 GLU109 CG −0.577 25.728 19.424 805 GLU109 CD 0.886 26.1 19.659 806 GLU109 OE1 1.612 25.244 20.147 807 GLU109 OE2 1.22 27.255 19.442 808 PHE110 N −0.686 22.572 15.873 809 PHE110 CA −0.511 21.269 15.221 810 PHE110 C 0.017 21.468 13.798 811 PHE110 O −0.547 20.963 12.819 812 PHE110 CB −1.866 20.568 15.206 813 PHE110 CG −1.834 19.088 14.838 814 PHE110 CD1 −0.842 18.264 15.352 815 PHE110 CD2 −2.808 18.564 13.998 816 PHE110 CE1 −0.819 16.915 15.019 817 PHE110 CE2 −2.784 17.216 13.665 818 PHE110 CZ −1.79 16.392 14.176 819 THR111 N 1.103 22.217 13.706 820 THR111 CA 1.68 22.585 12.409 821 THR111 C 2.353 21.411 11.71 822 THR111 O 2.846 20.47 12.346 823 THR111 CB 2.745 23.65 12.628 824 THR111 OG1 3.887 23.019 13.193 825 THR111 CG2 2.27 24.756 13.561 826 VAL112 N 2.564 21.605 10.417 827 VAL112 CA 3.302 20.63 9.605 828 VAL112 C 4.802 20.712 9.887 829 VAL112 O 5.492 19.689 9.86 830 VAL112 CB 3.026 20.929 8.134 831 VAL112 CG1 3.819 20.01 7.21 832 VAL112 CG2 1.535 20.833 7.832 833 LYS113 N 5.227 21.845 10.425 834 LYS113 CA 6.608 22.003 10.884 835 LYS113 C 6.892 21.096 12.082 836 LYS113 O 7.864 20.332 12.044 837 LYS113 CB 6.795 23.456 11.298 838 LYS113 CG 8.168 23.696 11.914 839 LYS113 CD 8.232 25.064 12.582 840 LYS113 CE 7.189 25.184 13.692 841 LYS113 NZ 7.407 24.178 14.747 842 ARG114 N 5.945 21.013 13.008 843 ARG114 CA 6.085 20.094 14.141 844 ARG114 C 6.098 18.627 13.713 845 ARG114 O 7.034 17.912 14.09 846 ARG114 CB 4.916 20.313 15.096 847 ARG114 CG 4.939 19.283 16.22 848 ARG114 CD 3.721 19.388 17.131 849 ARG114 NE 3.696 20.666 17.858 850 ARG114 CZ 4.078 20.792 19.131 851 ARG114 NH1 3.903 21.953 19.766 852 ARG114 NH2 4.537 19.73 19.798 853 MET115 N 5.28 18.265 12.737 854 MET115 CA 5.23 16.862 12.311 855 MET115 C 6.438 16.467 11.456 856 MET115 O 6.99 15.378 11.662 857 MET115 CB 3.926 16.642 11.555 858 MET115 CG 2.739 16.915 12.474 859 MET115 SD 1.093 16.637 11.78 860 MET115 CE 1.057 17.94 10.532 861 LYS116 N 7.027 17.445 10.787 862 LYS116 CA 8.25 17.222 10.01 863 LYS116 C 9.492 17.156 10.902 864 LYS116 O 10.434 16.413 10.59 865 LYS116 CB 8.372 18.392 9.042 866 LYS116 CG 9.635 18.337 8.194 867 LYS116 CD 9.738 19.592 7.338 868 LYS116 CE 9.703 20.841 8.213 869 LYS116 NZ 9.753 22.063 7.395 870 ALA117 N 9.404 17.748 12.084 871 ALA117 CA 10.49 17.663 13.066 872 ALA117 C 10.354 16.43 13.962 873 ALA117 O 11.331 16 14.587 874 ALA117 CB 10.469 18.924 13.922 875 LEU118 N 9.185 15.81 13.933 876 LEU118 CA 8.975 14.544 14.64 877 LEU118 C 9.351 13.359 13.76 878 LEU118 O 9.591 12.26 14.275 879 LEU118 CB 7.512 14.434 15.05 880 LEU118 CG 7.153 15.474 16.104 881 LEU118 CD1 5.654 15.48 16.372 882 LEU118 CD2 7.934 15.246 17.393 883 GLN119 N 9.563 13.632 12.483 884 GLN119 CA 10.052 12.633 11.518 885 GLN119 C 11.263 11.797 11.989 886 GLN119 O 11.09 10.573 12.041 887 GLN119 CB 10.378 13.373 10.227 888 GLN119 CG 10.944 12.471 9.144 889 GLN119 CD 11.394 13.351 7.985 890 GLN119 OE1 11.701 12.857 6.894 891 GLN119 NE2 11.444 14.647 8.243 892 PRO120 N 12.388 12.361 12.439 893 PRO120 CA 13.485 11.486 12.885 894 PRO120 C 13.211 10.732 14.195 895 PRO120 O 13.761 9.639 14.381 896 PRO120 CB 14.672 12.386 13.05 897 PRO120 CG 14.237 13.832 12.892 898 PRO120 CD 12.764 13.785 12.533 899 ARG121 N 12.229 11.159 14.974 900 ARG121 CA 11.917 10.438 16.203 901 ARG121 C 11.02 9.25 15.868 902 ARG121 O 11.331 8.136 16.303 903 ARG121 CB 11.218 11.385 17.174 904 ARG121 CG 11.741 11.209 18.597 905 ARG121 CD 11.481 9.812 19.149 906 ARG121 NE 12.184 9.613 20.424 907 ARG121 CZ 12.714 8.443 20.784 908 ARG121 NH1 13.415 8.346 21.915 909 ARG121 NH2 12.601 7.386 19.977 910 ILE122 N 10.18 9.421 14.857 911 ILE122 CA 9.316 8.332 14.381 912 ILE122 C 10.135 7.27 13.656 913 ILE122 O 9.975 6.073 13.928 914 ILE122 CB 8.309 8.918 13.396 915 ILE122 CG1 7.456 9.995 14.052 916 ILE122 CG2 7.422 7.825 12.807 917 ILE122 CD1 6.509 10.636 13.044 918 GLN123 N 11.179 7.724 12.982 919 GLN123 CA 12.088 6.827 12.269 920 GLN123 C 12.914 5.997 13.245 921 GLN123 O 12.897 4.76 13.156 922 GLN123 CB 12.989 7.717 11.423 923 GLN123 CG 13.978 6.941 10.567 924 GLN123 CD 14.72 7.939 9.684 925 GLN123 OE1 15.954 7.96 9.626 926 GLN123 NE2 13.946 8.8 9.044 927 GLN124 N 13.295 6.633 14.34 928 GLN124 CA 14.049 5.959 15.394 929 GLN124 C 13.184 4.947 16.144 930 GLN124 O 13.621 3.798 16.284 931 GLN124 CB 14.544 7.04 16.345 932 GLN124 CG 15.429 6.495 17.455 933 GLN124 CD 15.912 7.668 18.3 934 GLN124 OE1 16.786 7.524 19.162 935 GLN124 NE2 15.357 8.832 18.008 936 ILE125 N 11.904 5.252 16.299 937 ILE125 CA 10.961 4.328 16.947 938 ILE125 C 10.716 3.072 16.112 939 ILE125 O 10.861 1.961 16.642 940 ILE125 CB 9.638 5.064 17.148 941 ILE125 CG1 9.792 6.214 18.13 942 ILE125 CG2 8.543 4.122 17.628 943 ILE125 CD1 8.487 6.984 18.275 944 VAL126 N 10.649 3.229 14.797 945 VAL126 CA 10.44 2.064 13.928 946 VAL126 C 11.693 1.2 13.87 947 VAL126 O 11.603 −0.021 14.062 948 VAL126 CB 10.119 2.529 12.513 949 VAL126 CG1 9.754 1.334 11.641 950 VAL126 CG2 8.988 3.544 12.503 951 ASP127 N 12.843 1.855 13.909 952 ASP127 CA 14.121 1.141 13.889 953 ASP127 C 14.314 0.34 15.17 954 ASP127 O 14.537 −0.876 15.088 955 ASP127 CB 15.258 2.153 13.769 956 ASP127 CG 15.158 2.967 12.481 957 ASP127 OD1 15.632 4.097 12.49 958 ASP127 OD2 14.686 2.426 11.489 959 GLU128 N 13.903 0.919 16.288 960 GLU128 CA 14.048 0.26 17.589 961 GLU128 C 13.094 −0.915 17.762 962 GLU128 O 13.527 −1.952 18.281 963 GLU128 CB 13.764 1.281 18.684 964 GLU128 CG 14.807 2.39 18.707 965 GLU128 CD 14.367 3.489 19.668 966 GLU128 OE1 13.584 4.333 19.247 967 GLU128 OE2 14.794 3.452 20.812 968 HIS129 N 11.934 −0.861 17.128 969 HIS129 CA 11.002 −1.985 17.237 970 HIS129 C 11.411 −3.142 16.333 971 HIS129 O 11.344 −4.297 16.772 972 HIS129 CB 9.592 −1.533 16.885 973 HIS129 CG 8.963 −0.57 17.87 974 HIS129 ND1 7.942 0.266 17.612 975 HIS129 CD2 9.3 −0.394 19.192 976 HIS129 CE1 7.647 0.969 18.724 977 HIS129 NE2 8.488 0.561 19.701 978 ILE130 N 12.061 −2.848 15.218 979 ILE130 CA 12.564 −3.95 14.394 980 ILE130 C 13.768 −4.577 15.089 981 ILE130 O 13.69 −5.756 15.459 982 ILE130 CB 12.968 −3.449 13.012 983 ILE130 CG1 11.841 −2.659 12.36 984 ILE130 CG2 13.341 −4.632 12.125 985 ILE130 CD1 12.258 −2.117 10.996 986 ASP131 N 14.65 −3.712 15.575 987 ASP131 CA 15.874 −4.12 16.283 988 ASP131 C 15.604 −5.033 17.473 989 ASP131 O 15.932 −6.226 17.435 990 ASP131 CB 16.565 −2.874 16.84 991 ASP131 CG 17.175 −1.999 15.749 992 ASP131 OD1 17.222 −0.791 15.952 993 ASP131 OD2 17.743 −2.564 14.826 994 ALA132 N 14.882 −4.505 18.448 995 ALA132 CA 14.708 −5.198 19.727 996 ALA132 C 13.582 −6.228 19.763 997 ALA132 O 13.489 −6.983 20.738 998 ALA132 CB 14.465 −4.147 20.803 999 LEU133 N 12.752 −6.286 18.736 1000 LEU133 CA 11.712 −7.311 18.741 1001 LEU133 C 12.08 −8.444 17.798 1002 LEU133 O 12.567 −9.492 18.239 1003 LEU133 CB 10.366 −6.697 18.372 1004 LEU133 CG 9.925 −5.686 19.427 1005 LEU133 CD1 8.679 −4.93 18.987 1006 LEU133 CD2 9.698 −6.366 20.773 1007 LEU134 N 11.901 −8.215 16.511 1008 LEU134 CA 12.139 −9.288 15.539 1009 LEU134 C 12.895 −8.777 14.32 1010 LEU134 O 12.319 −8.632 13.237 1011 LEU134 CB 10.808 −9.885 15.087 1012 LEU134 CG 10.481 −11.234 15.731 1013 LEU134 CD1 11.66 −12.193 15.635 1014 LEU134 CD2 9.997 −11.115 17.173 1015 ALA135 N 14.194 −8.586 14.486 1016 ALA135 CA 15.038 −8.142 13.371 1017 ALA135 C 15.606 −9.293 12.538 1018 ALA135 O 16.184 −9.051 11.472 1019 ALA135 CB 16.193 −7.323 13.935 1020 GLY136 N 15.402 −10.522 12.984 1021 GLY136 CA 15.957 −11.679 12.272 1022 GLY136 C 14.865 −12.524 11.62 1023 GLY136 O 14.069 −12.022 10.829 1024 PRO137 N 14.906 −13.813 11.903 1025 PRO137 CA 13.938 −14.772 11.353 1026 PRO137 C 12.589 −14.709 12.067 1027 PRO137 O 12.21 −13.679 12.637 1028 PRO137 CB 14.573 −16.111 11.568 1029 PRO137 CG 15.748 −15.953 12.524 1030 PRO137 CD 15.899 −14.46 12.763 1031 LYS138 N 11.86 −15.811 11.945 1032 LYS138 CA 10.575 −16.043 12.639 1033 LYS138 C 9.412 −15.227 12.069 1034 LYS138 O 9.605 −14.143 11.508 1035 LYS138 CB 10.733 −15.765 14.135 1036 LYS138 CG 11.795 −16.66 14.765 1037 LYS138 CD 12.022 −16.303 16.23 1038 LYS138 CE 13.155 −17.128 16.829 1039 LYS138 NZ 12.859 −18.567 16.746 1040 PRO139 N 8.256 −15.868 12.022 1041 PRO139 CA 6.996 −15.155 11.797 1042 PRO139 C 6.612 −14.289 12.995 1043 PRO139 O 6.167 −14.795 14.031 1044 PRO139 CB 5.979 −16.232 11.583 1045 PRO139 CG 6.595 −17.575 11.948 1046 PRO139 CD 8.04 −17.286 12.322 1047 ALA140 N 6.749 −12.987 12.824 1048 ALA140 CA 6.355 −12.036 13.868 1049 ALA140 C 5.006 −11.411 13.552 1050 ALA140 O 4.591 −11.359 12.391 1051 ALA140 CB 7.395 −10.931 13.953 1052 ASP141 N 4.297 −10.989 14.582 1053 ASP141 CA 3.051 −10.264 14.336 1054 ASP141 C 3.363 −8.779 14.165 1055 ASP141 O 3.466 −8.032 15.149 1056 ASP141 CB 2.073 −10.492 15.481 1057 ASP141 CG 0.741 −9.84 15.132 1058 ASP141 OD1 0.583 −8.673 15.465 1059 ASP141 OD2 0.016 −10.426 14.338 1060 LEU142 N 3.261 −8.332 12.923 1061 LEU142 CA 3.692 −6.983 12.541 1062 LEU142 C 2.753 −5.893 13.048 1063 LEU142 O 3.232 −4.806 13.4 1064 LEU142 CB 3.78 −6.96 11.012 1065 LEU142 CG 4.336 −5.66 10.428 1066 LEU142 CD1 5.227 −5.944 9.225 1067 LEU142 CD2 3.241 −4.659 10.063 1068 VAL143 N 1.509 −6.243 13.329 1069 VAL143 CA 0.585 −5.247 13.868 1070 VAL143 C 0.992 −4.867 15.287 1071 VAL143 O 1.481 −3.748 15.477 1072 VAL143 CB −0.829 −5.809 13.859 1073 VAL143 CG1 −1.823 −4.772 14.374 1074 VAL143 CG2 −1.212 −6.26 12.457 1075 GLN144 N 1.184 −5.874 16.119 1076 GLN144 CA 1.491 −5.656 17.535 1077 GLN144 C 2.945 −5.249 17.797 1078 GLN144 O 3.212 −4.566 18.791 1079 GLN144 CB 1.203 −6.982 18.23 1080 GLN144 CG 1.485 −6.966 19.726 1081 GLN144 CD 1.232 −8.364 20.277 1082 GLN144 OE1 1.815 −8.777 21.285 1083 GLN144 NE2 0.374 −9.091 19.582 1084 ALA145 N 3.842 −5.539 16.87 1085 ALA145 CA 5.246 −5.186 17.088 1086 ALA145 C 5.672 −3.884 16.412 1087 ALA145 O 6.594 −3.211 16.888 1088 ALA145 CB 6.108 −6.329 16.561 1089 LEU146 N 4.988 −3.499 15.349 1090 LEU146 CA 5.424 −2.324 14.588 1091 LEU146 C 4.294 −1.342 14.317 1092 LEU146 O 4.308 −0.207 14.815 1093 LEU146 CB 5.964 −2.825 13.252 1094 LEU146 CG 7.225 −3.659 13.433 1095 LEU146 CD1 7.467 −4.589 12.254 1096 LEU146 CD2 8.432 −2.772 13.705 1097 SER147 N 3.245 −1.868 13.71 1098 SER147 CA 2.169 −1.053 13.134 1099 SER147 C 1.274 −0.369 14.164 1100 SER147 O 0.727 0.708 13.911 1101 SER147 CB 1.325 −2.001 12.301 1102 SER147 OG 0.198 −1.277 11.856 1103 LEU148 N 1.174 −0.97 15.331 1104 LEU148 CA 0.484 −0.358 16.464 1105 LEU148 C 1.433 0.51 17.316 1106 LEU148 O 1.132 1.707 17.436 1107 LEU148 CB −0.204 −1.475 17.259 1108 LEU148 CG −1.13 −0.987 18.372 1109 LEU148 CD1 −2.317 −1.93 18.53 1110 LEU148 CD2 −0.404 −0.81 19.703 1111 PRO149 N 2.553 0.008 17.848 1112 PRO149 CA 3.299 0.829 18.809 1113 PRO149 C 3.987 2.052 18.203 1114 PRO149 O 4.043 3.07 18.9 1115 PRO149 CB 4.31 −0.075 19.439 1116 PRO149 CG 4.261 −1.43 18.764 1117 PRO149 CD 3.122 −1.352 17.766 1118 VAL150 N 4.316 2.051 16.918 1119 VAL150 CA 4.873 3.276 16.323 1120 VAL150 C 3.867 4.442 16.385 1121 VAL150 O 4.127 5.334 17.201 1122 VAL150 CB 5.399 3.041 14.905 1123 VAL150 CG1 5.892 4.345 14.284 1124 VAL150 CG2 6.514 2.004 14.891 1125 PRO151 N 2.695 4.396 15.75 1126 PRO151 CA 1.816 5.574 15.799 1127 PRO151 C 1.187 5.843 17.167 1128 PRO151 O 1.009 7.014 17.532 1129 PRO151 CB 0.742 5.307 14.799 1130 PRO151 CG 0.88 3.895 14.266 1131 PRO151 CD 2.136 3.336 14.896 1132 SER152 N 1.059 4.817 17.993 1133 SER152 CA 0.504 5.027 19.326 1134 SER152 C 1.506 5.73 20.241 1135 SER152 O 1.136 6.724 20.879 1136 SER152 CB 0.117 3.671 19.898 1137 SER152 OG −0.849 3.09 19.031 1138 LEU153 N 2.785 5.428 20.079 1139 LEU153 CA 3.817 6.088 20.883 1140 LEU153 C 4.17 7.458 20.312 1141 LEU153 O 4.34 8.406 21.091 1142 LEU153 CB 5.06 5.206 20.899 1143 LEU153 CG 6.168 5.789 21.769 1144 LEU153 CD1 5.708 5.934 23.216 1145 LEU153 CD2 7.424 4.928 21.689 1146 VAL154 N 3.995 7.622 19.009 1147 VAL154 CA 4.232 8.925 18.383 1148 VAL154 C 3.185 9.944 18.813 1149 VAL154 O 3.566 11.036 19.256 1150 VAL154 CB 4.205 8.777 16.864 1151 VAL154 CG1 4.148 10.134 16.169 1152 VAL154 CG2 5.402 7.976 16.368 1153 ILE155 N 1.945 9.513 18.977 1154 ILE155 CA 0.935 10.467 19.431 1155 ILE155 C 0.932 10.621 20.956 1156 ILE155 O 0.6 11.709 21.446 1157 ILE155 CB −0.433 10.065 18.902 1158 ILE155 CG1 −1.405 11.218 19.089 1159 ILE155 CG2 −0.956 8.814 19.593 1160 ILE155 CD1 −0.954 12.46 18.327 1161 CYS156 N 1.569 9.697 21.66 1162 CYS156 CA 1.787 9.891 23.093 1163 CYS156 C 2.835 10.973 23.317 1164 CYS156 O 2.551 11.93 24.047 1165 CYS156 CB 2.261 8.59 23.732 1166 CYS156 SG 1.018 7.295 23.935 1167 GLU157 N 3.838 11.005 22.454 1168 GLU157 CA 4.902 12.011 22.559 1169 GLU157 C 4.512 13.364 21.957 1170 GLU157 O 5.08 14.393 22.339 1171 GLU157 CB 6.109 11.474 21.801 1172 GLU157 CG 6.57 10.14 22.372 1173 GLU157 CD 7.588 9.499 21.434 1174 GLU157 OE1 8.764 9.522 21.767 1175 GLU157 OE2 7.162 8.932 20.437 1176 LEU158 N 3.5 13.37 21.107 1177 LEU158 CA 3.042 14.616 20.492 1178 LEU158 C 2.03 15.308 21.401 1179 LEU158 O 2.089 16.535 21.586 1180 LEU158 CB 2.412 14.232 19.149 1181 LEU158 CG 2.171 15.389 18.175 1182 LEU158 CD1 2.086 14.868 16.745 1183 LEU158 CD2 0.93 16.213 18.504 1184 LEU159 N 1.211 14.518 22.072 1185 LEU159 CA 0.183 15.099 22.929 1186 LEU159 C 0.747 15.411 24.309 1187 LEU159 O 0.832 16.594 24.66 1188 LEU159 CB −0.979 14.12 23.044 1189 LEU159 CG −2.24 14.827 23.524 1190 LEU159 CD1 −2.637 15.915 22.535 1191 LEU159 CD2 −3.385 13.84 23.707 1192 GLY160 N 1.347 14.417 24.943 1193 GLY160 CA 1.874 14.572 26.306 1194 GLY160 C 1.433 13.413 27.2 1195 GLY160 O 1.183 13.581 28.398 1196 VAL161 N 1.366 12.238 26.601 1197 VAL161 CA 0.869 11.037 27.284 1198 VAL161 C 2.006 10.078 27.635 1199 VAL161 O 2.715 9.58 26.752 1200 VAL161 CB −0.111 10.348 26.338 1201 VAL161 CG1 −0.763 9.125 26.976 1202 VAL161 CG2 −1.175 11.327 25.866 1203 PRO162 N 2.15 9.806 28.922 1204 PRO162 CA 3.13 8.826 29.4 1205 PRO162 C 2.838 7.41 28.901 1206 PRO162 O 1.68 7.021 28.689 1207 PRO162 CB 3.066 8.899 30.894 1208 PRO162 CG 1.972 9.874 31.302 1209 PRO162 CD 1.358 10.384 30.009 1210 TYR163 N 3.882 6.593 28.912 1211 TYR163 CA 3.793 5.209 28.41 1212 TYR163 C 3.104 4.257 29.393 1213 TYR163 O 2.72 3.153 29.002 1214 TYR163 CB 5.192 4.684 28.071 1215 TYR163 CG 6.111 4.367 29.254 1216 TYR163 CD1 6.895 5.363 29.826 1217 TYR163 CD2 6.181 3.067 29.743 1218 TYR163 CE1 7.726 5.066 30.898 1219 TYR163 CE2 7.011 2.768 30.815 1220 TYR163 CZ 7.78 3.77 31.392 1221 TYR163 OH 8.589 3.478 32.467 1222 SER164 N 2.806 4.747 30.589 1223 SER164 CA 2.007 3.992 31.561 1224 SER164 C 0.51 4.158 31.301 1225 SER164 O −0.319 3.566 32 1226 SER164 CB 2.303 4.538 32.952 1227 SER164 OG 1.766 5.854 33.017 1228 ASP165 N 0.173 5.056 30.389 1229 ASP165 CA −1.215 5.273 30.002 1230 ASP165 C −1.445 4.619 28.649 1231 ASP165 O −2.542 4.126 28.344 1232 ASP165 CB −1.463 6.775 29.944 1233 ASP165 CG −1.331 7.38 31.343 1234 ASP165 OD1 −2.358 7.592 31.968 1235 ASP165 OD2 −0.21 7.683 31.732 1236 HIS166 N −0.343 4.475 27.929 1237 HIS166 CA −0.299 3.666 26.71 1238 HIS166 C −0.63 2.223 27.09 1239 HIS166 O −0.515 1.86 28.266 1240 HIS166 CB 1.11 3.792 26.129 1241 HIS166 CG 1.387 3.018 24.856 1242 HIS166 ND1 0.916 3.295 23.626 1243 HIS166 CD2 2.174 1.896 24.741 1244 HIS166 CE1 1.378 2.374 22.756 1245 HIS166 NE2 2.156 1.51 23.445 1246 GLU167 N −1.239 1.506 26.155 1247 GLU167 CA −1.754 0.127 26.342 1248 GLU167 C −3.12 0.07 27.045 1249 GLU167 O −4.04 −0.549 26.498 1250 GLU167 CB −0.743 −0.752 27.077 1251 GLU167 CG 0.54 −0.919 26.271 1252 GLU167 CD 1.593 −1.638 27.107 1253 GLU167 OE1 1.207 −2.478 27.907 1254 GLU167 OE2 2.766 −1.386 26.875 1255 PHE168 N −3.329 0.846 28.097 1256 PHE168 CA −4.652 0.854 28.729 1257 PHE168 C −5.586 1.779 27.954 1258 PHE168 O −6.692 1.36 27.587 1259 PHE168 CB −4.53 1.304 30.179 1260 PHE168 CG −5.824 1.165 30.977 1261 PHE168 CD1 −6.696 0.117 30.709 1262 PHE168 CD2 −6.126 2.078 31.979 1263 PHE168 CE1 −7.875 −0.01 31.432 1264 PHE168 CE2 −7.305 1.951 32.703 1265 PHE168 CZ −8.18 0.908 32.428 1266 PHE169 N −5.016 2.851 27.423 1267 PHE169 CA −5.773 3.717 26.514 1268 PHE169 C −5.944 3.029 25.162 1269 PHE169 O −7.031 3.098 24.579 1270 PHE169 CB −5.003 5.024 26.316 1271 PHE169 CG −5.736 6.088 25.497 1272 PHE169 CD1 −6.361 7.145 26.147 1273 PHE169 CD2 −5.758 6.022 24.109 1274 PHE169 CE1 −7.037 8.111 25.413 1275 PHE169 CE2 −6.436 6.985 23.375 1276 PHE169 CZ −7.082 8.028 24.027 1277 GLN170 N −5.019 2.134 24.851 1278 GLN170 CA −5.042 1.407 23.584 1279 GLN170 C −6.148 0.359 23.564 1280 GLN170 O −6.903 0.281 22.588 1281 GLN170 CB −3.705 0.693 23.444 1282 GLN170 CG −3.611 −0.121 22.163 1283 GLN170 CD −3.411 0.818 20.985 1284 GLN170 OE1 −2.392 1.516 20.917 1285 GLN170 NE2 −4.408 0.885 20.125 1286 SER171 N −6.372 −0.281 24.698 1287 SER171 CA −7.424 −1.295 24.769 1288 SER171 C −8.811 −0.665 24.856 1289 SER171 O −9.706 −1.107 24.125 1290 SER171 CB −7.171 −2.209 25.967 1291 SER171 OG −7.132 −1.427 27.155 1292 CYS172 N −8.906 0.511 25.457 1293 CYS172 CA −10.204 1.184 25.525 1294 CYS172 C −10.569 1.806 24.18 1295 CYS172 O −11.691 1.593 23.703 1296 CYS172 CB −10.149 2.266 26.593 1297 CYS172 SG −9.817 1.701 28.277 1298 SER173 N −9.559 2.253 23.453 1299 SER173 CA −9.781 2.826 22.121 1300 SER173 C −9.949 1.765 21.033 1301 SER173 O −10.363 2.096 19.916 1302 SER173 CB −8.612 3.741 21.775 1303 SER173 OG −7.418 2.972 21.747 1304 SER174 N −9.687 0.509 21.356 1305 SER174 CA −10.001 −0.575 20.427 1306 SER174 C −11.395 −1.133 20.713 1307 SER174 O −12.128 −1.464 19.771 1308 SER174 CB −8.949 −1.668 20.566 1309 SER174 OG −7.691 −1.099 20.225 1310 ARG175 N −11.85 −0.974 21.949 1311 ARG175 CA −13.224 −1.373 22.294 1312 ARG175 C −14.226 −0.342 21.787 1313 ARG175 O −15.298 −0.72 21.298 1314 ARG175 CB −13.371 −1.514 23.805 1315 ARG175 CG −12.486 −2.622 24.36 1316 ARG175 CD −12.761 −2.871 25.837 1317 ARG175 NE −12.544 −1.662 26.646 1318 ARG175 CZ −13.06 −1.512 27.869 1319 ARG175 NH1 −12.78 −0.421 28.584 1320 ARG175 NH2 −13.816 −2.478 28.397 1321 MET176 N −13.739 0.876 21.611 1322 MET176 CA −14.516 1.951 20.985 1323 MET176 C −14.649 1.843 19.467 1324 MET176 O −15.284 2.71 18.855 1325 MET176 CB −13.806 3.259 21.259 1326 MET176 CG −13.86 3.657 22.721 1327 MET176 SD −13.036 5.228 23.009 1328 MET176 CE −13.493 5.972 21.425 1329 LEU177 N −14.036 0.846 18.853 1330 LEU177 CA −14.184 0.676 17.411 1331 LEU177 C −15.219 −0.388 17.059 1332 LEU177 O −15.593 −0.533 15.886 1333 LEU177 CB −12.836 0.29 16.827 1334 LEU177 CG −11.824 1.426 16.9 1335 LEU177 CD1 −10.568 1.035 16.135 1336 LEU177 CD2 −12.4 2.713 16.32 1337 SER178 N −15.706 −1.099 18.062 1338 SER178 CA −16.693 −2.149 17.793 1339 SER178 C −18.117 −1.673 18.08 1340 SER178 O −18.628 −1.797 19.2 1341 SER178 CB −16.344 −3.402 18.599 1342 SER178 OG −16.323 −3.095 19.988 1343 ARG179 N −18.765 −1.209 17.022 1344 ARG179 CA −20.148 −0.697 17.082 1345 ARG179 C −21.17 −1.832 17.192 1346 ARG179 O −21.743 −2.266 16.187 1347 ARG179 CB −20.4 0.027 15.768 1348 ARG179 CG −19.286 1.01 15.431 1349 ARG179 CD −19.098 1.114 13.922 1350 ARG179 NE −18.628 −0.179 13.39 1351 ARG179 CZ −19.337 −0.967 12.576 1352 ARG179 NH1 −18.903 −2.2 12.307 1353 ARG179 NH2 −20.546 −0.583 12.156 1354 GLU180 N −21.375 −2.313 18.405 1355 GLU180 CA −22.261 −3.454 18.627 1356 GLU180 C −23.553 −3.038 19.318 1357 GLU180 O −23.676 −1.923 19.833 1358 GLU180 CB −21.517 −4.449 19.508 1359 GLU180 CG −20.175 −4.838 18.899 1360 GLU180 CD −19.442 −5.795 19.828 1361 GLU180 OE1 −20.124 −6.592 20.457 1362 GLU180 OE2 −18.219 −5.763 19.834 1363 VAL181 N −24.492 −3.969 19.374 1364 VAL181 CA −25.72 −3.744 20.147 1365 VAL181 C −25.463 −4.084 21.616 1366 VAL181 O −26.012 −3.452 22.525 1367 VAL181 CB −26.823 −4.627 19.569 1368 VAL181 CG1 −28.119 −4.498 20.362 1369 VAL181 CG2 −27.062 −4.297 18.099 1370 THR182 N −24.438 −4.897 21.822 1371 THR182 CA −23.936 −5.215 23.166 1372 THR182 C −22.767 −4.302 23.547 1373 THR182 O −21.827 −4.739 24.222 1374 THR182 CB −23.459 −6.664 23.183 1375 THR182 OG1 −22.348 −6.789 22.302 1376 THR182 CG2 −24.551 −7.622 22.719 1377 ALA183 N −22.882 −3.025 23.214 1378 ALA183 CA −21.777 −2.062 23.352 1379 ALA183 C −21.585 −1.459 24.748 1380 ALA183 O −21.031 −0.36 24.855 1381 ALA183 CB −21.993 −0.933 22.352 1382 GLU184 N −21.832 −2.224 25.799 1383 GLU184 CA −21.706 −1.688 27.158 1384 GLU184 C −20.242 −1.537 27.568 1385 GLU184 O −19.888 −0.536 28.199 1386 GLU184 CB −22.415 −2.636 28.117 1387 GLU184 CG −22.387 −2.118 29.551 1388 GLU184 CD −23.145 −3.086 30.454 1389 GLU184 OE1 −22.924 −3.047 31.655 1390 GLU184 OE2 −23.953 −3.832 29.917 1391 GLU185 N −19.374 −2.344 26.978 1392 GLU185 CA −17.936 −2.201 27.244 1393 GLU185 C −17.307 −1.143 26.337 1394 GLU185 O −16.294 −0.542 26.708 1395 GLU185 CB −17.216 −3.541 27.073 1396 GLU185 CG −17.189 −4.388 28.351 1397 GLU185 CD −18.561 −4.959 28.705 1398 GLU185 OE1 −19.308 −5.237 27.774 1399 GLU185 OE2 −18.899 −4.953 29.879 1400 ARG186 N −18.044 −0.74 25.316 1401 ARG186 CA −17.607 0.339 24.434 1402 ARG186 C −18.008 1.676 25.049 1403 ARG186 O −17.213 2.624 25.056 1404 ARG186 CB −18.317 0.135 23.103 1405 ARG186 CG −17.936 1.175 22.064 1406 ARG186 CD −18.718 0.942 20.779 1407 ARG186 NE −18.234 1.82 19.708 1408 ARG186 CZ −19.002 2.705 19.073 1409 ARG186 NH1 −20.296 2.816 19.386 1410 ARG186 NH2 −18.474 3.477 18.122 1411 MET187 N −19.086 1.627 25.817 1412 MET187 CA −19.545 2.775 26.601 1413 MET187 C −18.651 2.978 27.82 1414 MET187 O −18.251 4.113 28.111 1415 MET187 CB −20.958 2.459 27.07 1416 MET187 CG −21.569 3.594 27.88 1417 MET187 SD −23.104 3.161 28.726 1418 MET187 CE −23.995 2.43 27.333 1419 THR188 N −18.13 1.874 28.332 1420 THR188 CA −17.174 1.925 29.44 1421 THR188 C −15.819 2.442 28.969 1422 THR188 O −15.237 3.307 29.634 1423 THR188 CB −17.013 0.516 30 1424 THR188 OG1 −18.274 0.091 30.499 1425 THR188 CG2 −16.011 0.473 31.149 1426 ALA189 N −15.477 2.139 27.726 1427 ALA189 CA −14.243 2.652 27.133 1428 ALA189 C −14.333 4.147 26.838 1429 ALA189 O −13.403 4.884 27.192 1430 ALA189 CB −13.996 1.884 25.844 1431 PHE190 N −15.518 4.605 26.461 1432 PHE190 CA −15.762 6.042 26.26 1433 PHE190 C −15.632 6.81 27.572 1434 PHE190 O −14.815 7.736 27.678 1435 PHE190 CB −17.194 6.258 25.773 1436 PHE190 CG −17.566 5.772 24.375 1437 PHE190 CD1 −16.652 5.837 23.334 1438 PHE190 CD2 −18.848 5.293 24.139 1439 PHE190 CE1 −17.013 5.406 22.063 1440 PHE190 CE2 −19.208 4.861 22.871 1441 PHE190 CZ −18.291 4.917 21.832 1442 GLU191 N −16.281 6.297 28.605 1443 GLU191 CA −16.292 6.969 29.908 1444 GLU191 C −14.921 6.971 30.575 1445 GLU191 O −14.432 8.05 30.936 1446 GLU191 CB −17.28 6.233 30.802 1447 GLU191 CG −17.348 6.853 32.193 1448 GLU191 CD −18.28 6.023 33.069 1449 GLU191 OE1 −18.424 4.843 32.777 1450 GLU191 OE2 −18.894 6.597 33.957 1451 SER192 N −14.202 5.866 30.453 1452 SER192 CA −12.877 5.76 31.071 1453 SER192 C −11.82 6.545 30.301 1454 SER192 O −10.892 7.077 30.921 1455 SER192 CB −12.467 4.291 31.126 1456 SER192 OG −12.37 3.801 29.792 1457 LEU193 N −12.082 6.827 29.036 1458 LEU193 CA −11.152 7.64 28.256 1459 LEU193 C −11.441 9.127 28.435 1460 LEU193 O −10.514 9.943 28.377 1461 LEU193 CB −11.243 7.218 26.798 1462 LEU193 CG −9.95 6.547 26.343 1463 LEU193 CD1 −9.372 5.603 27.391 1464 LEU193 CD2 −10.117 5.842 25.003 1465 GLU194 N −12.63 9.438 28.923 1466 GLU194 CA −12.93 10.811 29.327 1467 GLU194 C −12.368 11.096 30.711 1468 GLU194 O −11.82 12.182 30.938 1469 GLU194 CB −14.437 11.017 29.337 1470 GLU194 CG −14.97 11.164 27.922 1471 GLU194 CD −14.35 12.405 27.287 1472 GLU194 OE1 −13.524 12.237 26.403 1473 GLU194 OE2 −14.826 13.487 27.596 1474 ASN195 N −12.26 10.058 31.523 1475 ASN195 CA −11.645 10.215 32.844 1476 ASN195 C −10.126 10.268 32.715 1477 ASN195 O −9.479 11.112 33.356 1478 ASN195 CB −12.076 9.054 33.736 1479 ASN195 CG −13.582 9.104 34.008 1480 ASN195 OD1 −14.272 8.076 33.958 1481 ASN195 ND2 −14.076 10.3 34.287 1482 TYR196 N −9.64 9.613 31.673 1483 TYR196 CA −8.236 9.692 31.267 1484 TYR196 C −7.88 11.104 30.817 1485 TYR196 O −6.953 11.707 31.371 1486 TYR196 CB −8.065 8.757 30.078 1487 TYR196 CG −7.054 7.633 30.253 1488 TYR196 CD1 −5.806 7.739 29.658 1489 TYR196 CD2 −7.39 6.5 30.982 1490 TYR196 CE1 −4.884 6.711 29.799 1491 TYR196 CE2 −6.466 5.472 31.126 1492 TYR196 CZ −5.216 5.581 30.532 1493 TYR196 OH −4.301 4.56 30.661 1494 LEU197 N −8.76 11.708 30.032 1495 LEU197 CA −8.543 13.084 29.57 1496 LEU197 C −8.718 14.131 30.663 1497 LEU197 O −7.966 15.11 30.658 1498 LEU197 CB −9.53 13.394 28.458 1499 LEU197 CG −9.196 12.63 27.188 1500 LEU197 CD1 −10.305 12.816 26.168 1501 LEU197 CD2 −7.852 13.072 26.619 1502 ASP198 N −9.49 13.83 31.695 1503 ASP198 CA −9.6 14.749 32.834 1504 ASP198 C −8.257 14.841 33.551 1505 ASP198 O −7.698 15.938 33.694 1506 ASP198 CB −10.627 14.209 33.829 1507 ASP198 CG −12.016 14.052 33.214 1508 ASP198 OD1 −12.428 14.949 32.492 1509 ASP198 OD2 −12.706 13.123 33.627 1510 GLU199 N −7.629 13.685 33.696 1511 GLU199 CA −6.334 13.598 34.368 1512 GLU199 C −5.221 14.182 33.506 1513 GLU199 O −4.521 15.091 33.965 1514 GLU199 CB −6.053 12.121 34.608 1515 GLU199 CG −7.157 11.486 35.444 1516 GLU199 CD −7.084 9.966 35.341 1517 GLU199 OE1 −7.502 9.312 36.287 1518 GLU199 OE2 −6.717 9.484 34.277 1519 LEU200 N −5.26 13.885 32.219 1520 LEU200 CA −4.209 14.315 31.289 1521 LEU200 C −4.22 15.821 31.02 1522 LEU200 O −3.168 16.467 31.128 1523 LEU200 CB −4.459 13.57 29.982 1524 LEU200 CG −3.421 13.888 28.914 1525 LEU200 CD1 −2.036 13.415 29.338 1526 LEU200 CD2 −3.815 13.251 27.587 1527 VAL201 N −5.406 16.402 30.938 1528 VAL201 CA −5.521 17.832 30.642 1529 VAL201 C −5.196 18.69 31.859 1530 VAL201 O −4.491 19.7 31.709 1531 VAL201 CB −6.945 18.094 30.153 1532 VAL201 CG1 −7.324 19.57 30.184 1533 VAL201 CG2 −7.159 17.508 28.761 1534 THR202 N −5.431 18.149 33.045 1535 THR202 CA −5.103 18.884 34.267 1536 THR202 C −3.643 18.677 34.654 1537 THR202 O −2.981 19.624 35.101 1538 THR202 CB −6.02 18.391 35.378 1539 THR202 OG1 −7.359 18.589 34.945 1540 THR202 CG2 −5.814 19.173 36.671 1541 LYS203 N −3.08 17.579 34.182 1542 LYS203 CA −1.672 17.284 34.434 1543 LYS203 C −0.755 18.113 33.539 1544 LYS203 O 0.305 18.539 34.015 1545 LYS203 CB −1.474 15.79 34.209 1546 LYS203 CG −0.041 15.331 34.439 1547 LYS203 CD 0.025 13.812 34.569 1548 LYS203 CE −0.557 13.099 33.352 1549 LYS203 NZ 0.265 13.325 32.154 1550 LYS204 N −1.256 18.545 32.391 1551 LYS204 CA −0.505 19.493 31.553 1552 LYS204 C −0.788 20.954 31.896 1553 LYS204 O −0.031 21.846 31.499 1554 LYS204 CB −0.821 19.218 30.092 1555 LYS204 CG −0.076 17.965 29.664 1556 LYS204 CD 1.425 18.213 29.729 1557 LYS204 CE 2.202 16.906 29.69 1558 LYS204 NZ 1.918 16.115 30.896 1559 GLU205 N −1.781 21.179 32.741 1560 GLU205 CA −2.033 22.524 33.264 1561 GLU205 C −1.215 22.777 34.526 1562 GLU205 O −1.027 23.931 34.927 1563 GLU205 CB −3.518 22.66 33.57 1564 GLU205 CG −4.309 22.726 32.273 1565 GLU205 CD −5.788 22.444 32.51 1566 GLU205 OE1 −6.547 22.661 31.573 1567 GLU205 OE2 −6.093 21.798 33.504 1568 ALA206 N −0.712 21.707 35.12 1569 ALA206 CA 0.208 21.844 36.249 1570 ALA206 C 1.649 21.772 35.756 1571 ALA206 O 2.464 22.661 36.034 1572 ALA206 CB −0.059 20.711 37.233 1573 ASN207 N 1.92 20.765 34.945 1574 ASN207 CA 3.253 20.587 34.366 1575 ASN207 C 3.307 21.204 32.976 1576 ASN207 O 2.922 20.574 31.982 1577 ASN207 CB 3.568 19.096 34.27 1578 ASN207 CG 3.565 18.441 35.65 1579 ASN207 OD1 4.361 18.793 36.527 1580 ASN207 ND2 2.641 17.515 35.835 1581 ALA208 N 3.786 22.435 32.932 1582 ALA208 CA 3.924 23.166 31.668 1583 ALA208 C 5.045 22.601 30.795 1584 ALA208 O 6.235 22.809 31.052 1585 ALA208 CB 4.202 24.631 31.982 1586 THR209 N 4.636 21.874 29.77 1587 THR209 CA 5.581 21.251 28.834 1588 THR209 C 5.499 21.873 27.446 1589 THR209 O 4.665 22.737 27.168 1590 THR209 CB 5.253 19.774 28.706 1591 THR209 OG1 3.938 19.693 28.18 1592 THR209 CG2 5.296 19.059 30.052 1593 GLU210 N 6.34 21.363 26.562 1594 GLU210 CA 6.411 21.861 25.18 1595 GLU210 C 5.542 21.076 24.19 1596 GLU210 O 5.586 21.361 22.989 1597 GLU210 CB 7.861 21.818 24.692 1598 GLU210 CG 8.791 22.752 25.467 1599 GLU210 CD 9.689 21.967 26.424 1600 GLU210 OE1 9.258 20.906 26.861 1601 GLU210 OE2 10.778 22.442 26.707 1602 ASP211 N 4.786 20.097 24.662 1603 ASP211 CA 3.977 19.273 23.746 1604 ASP211 C 2.732 20.003 23.249 1605 ASP211 O 2.331 21.027 23.817 1606 ASP211 CB 3.605 17.956 24.418 1607 ASP211 CG 2.942 18.154 25.781 1608 ASP211 OD1 2.12 19.054 25.925 1609 ASP211 OD2 3.336 17.427 26.681 1610 ASP212 N 2.034 19.382 22.309 1611 ASP212 CA 0.906 20.044 21.64 1612 ASP212 C −0.365 20.067 22.496 1613 ASP212 O −1.223 20.928 22.262 1614 ASP212 CB 0.653 19.331 20.312 1615 ASP212 CG −0.355 20.078 19.435 1616 ASP212 OD1 −0.505 21.277 19.623 1617 ASP212 OD2 −1.022 19.415 18.653 1618 LEU213 N −0.398 19.318 23.589 1619 LEU213 CA −1.529 19.451 24.508 1620 LEU213 C −1.474 20.819 25.18 1621 LEU213 O −2.372 21.628 24.907 1622 LEU213 CB −1.498 18.346 25.557 1623 LEU213 CG −2.75 18.365 26.427 1624 LEU213 CD1 −4.011 18.334 25.574 1625 LEU213 CD2 −2.751 17.202 27.41 1626 LEU214 N −0.306 21.193 25.691 1627 LEU214 CA −0.187 22.52 26.307 1628 LEU214 C −0.094 23.607 25.241 1629 LEU214 O −0.613 24.711 25.445 1630 LEU214 CB 1.034 22.608 27.21 1631 LEU214 CG 0.987 23.94 27.954 1632 LEU214 CD1 −0.18 23.972 28.934 1633 LEU214 CD2 2.288 24.25 28.671 1634 GLY215 N 0.35 23.222 24.056 1635 GLY215 CA 0.292 24.093 22.882 1636 GLY215 C −1.121 24.618 22.629 1637 GLY215 O −1.327 25.838 22.586 1638 ARG216 N −2.103 23.733 22.582 1639 ARG216 CA −3.473 24.198 22.351 1640 ARG216 C −4.154 24.727 23.616 1641 ARG216 O −5.077 25.542 23.506 1642 ARG216 CB −4.3 23.068 21.765 1643 ARG216 CG −3.636 22.456 20.539 1644 ARG216 CD −4.555 21.415 19.912 1645 ARG216 NE −5.402 20.812 20.953 1646 ARG216 CZ −5.073 19.756 21.699 1647 ARG216 NH1 −3.937 19.093 21.471 1648 ARG216 NH2 −5.905 19.342 22.652 1649 GLN217 N −3.583 24.445 24.776 1650 GLN217 CA −4.102 25.002 26.031 1651 GLN217 C −3.624 26.435 26.28 1652 GLN217 O −4.198 27.131 27.125 1653 GLN217 CB −3.648 24.109 27.181 1654 GLN217 CG −4.235 22.711 27.043 1655 GLN217 CD −3.691 21.772 28.114 1656 GLN217 OE1 −2.544 21.311 28.052 1657 GLN217 NE2 −4.552 21.446 29.059 1658 ILE218 N −2.608 26.875 25.551 1659 ILE218 CA −2.179 28.276 25.625 1660 ILE218 C −2.638 29.086 24.409 1661 ILE218 O −2.242 30.25 24.267 1662 ILE218 CB −0.66 28.352 25.774 1663 ILE218 CG1 0.061 27.755 24.572 1664 ILE218 CG2 −0.211 27.665 27.059 1665 ILE218 CD1 1.574 27.781 24.754 1666 LEU219 N −3.431 28.478 23.538 1667 LEU219 CA −3.953 29.194 22.365 1668 LEU219 C −4.858 30.352 22.75 1669 LEU219 O −5.716 30.229 23.629 1670 LEU219 CB −4.756 28.24 21.493 1671 LEU219 CG −3.859 27.411 20.59 1672 LEU219 CD1 −4.674 26.345 19.873 1673 LEU219 CD2 −3.135 28.304 19.588 1674 LYS220 N −4.667 31.454 22.047 1675 LYS220 CA −5.484 32.654 22.234 1676 LYS220 C −5.341 33.562 21.012 1677 LYS220 O −4.556 34.519 21.016 1678 LYS220 CB −5.01 33.38 23.489 1679 LYS220 CG −5.91 34.56 23.842 1680 LYS220 CD −5.389 35.289 25.074 1681 LYS220 CE −6.258 36.494 25.418 1682 LYS220 NZ −5.74 37.191 26.607 1683 GLN221 N −6.048 33.217 19.951 1684 GLN221 CA −5.987 34.031 18.732 1685 GLN221 C −6.904 35.234 18.892 1686 GLN221 O −7.981 35.112 19.48 1687 GLN221 CB −6.389 33.179 17.535 1688 GLN221 CG −5.417 32.015 17.369 1689 GLN221 CD −5.828 31.088 16.227 1690 GLN221 OE1 −7.019 30.872 15.966 1691 GLN221 NE2 −4.823 30.508 15.595 1692 ARG222 N −6.553 36.345 18.268 1693 ARG222 CA −7.309 37.589 18.486 1694 ARG222 C −8.774 37.467 18.066 1695 ARG222 O −9.669 37.72 18.877 1696 ARG222 CB −6.649 38.705 17.685 1697 ARG222 CG −7.362 40.034 17.911 1698 ARG222 CD −6.787 41.134 17.026 1699 ARG222 NE −7.505 42.401 17.232 1700 ARG222 CZ −8.368 42.909 16.349 1701 ARG222 NH1 −8.626 42.255 15.213 1702 ARG222 NH2 −8.98 44.068 16.604 1703 GLU223 N −9.009 36.864 16.912 1704 GLU223 CA −10.382 36.702 16.426 1705 GLU223 C −10.985 35.329 16.735 1706 GLU223 O −12.083 35.029 16.257 1707 GLU223 CB −10.395 36.959 14.926 1708 GLU223 CG −9.977 38.396 14.634 1709 GLU223 CD −9.946 38.649 13.13 1710 GLU223 OE1 −9.041 39.35 12.701 1711 GLU223 OE2 −10.749 38.047 12.434 1712 SER224 N −10.28 34.5 17.488 1713 SER224 CA −10.803 33.162 17.775 1714 SER224 C −11.036 32.957 19.266 1715 SER224 O −11.791 32.067 19.67 1716 SER224 CB −9.789 32.13 17.308 1717 SER224 OG −9.509 32.374 15.941 1718 GLY225 N −10.409 33.796 20.069 1719 GLY225 CA −10.436 33.613 21.517 1720 GLY225 C −9.539 32.435 21.884 1721 GLY225 O −8.577 32.109 21.174 1722 GLU226 N −9.863 31.807 22.998 1723 GLU226 CA −9.128 30.613 23.422 1724 GLU226 C −10.067 29.417 23.535 1725 GLU226 O −11.22 29.553 23.963 1726 GLU226 CB −8.424 30.894 24.745 1727 GLU226 CG −9.368 31.316 25.86 1728 GLU226 CD −8.548 31.714 27.084 1729 GLU226 OE1 −8.364 30.873 27.952 1730 GLU226 OE2 −8.166 32.875 27.147 1731 ALA227 N −9.567 28.262 23.127 1732 ALA227 CA −10.372 27.035 23.163 1733 ALA227 C −10.725 26.657 24.598 1734 ALA227 O −9.859 26.603 25.477 1735 ALA227 CB −9.587 25.904 22.508 1736 ASP228 N −12.009 26.456 24.833 1737 ASP228 CA −12.471 26.085 26.175 1738 ASP228 C −12.163 24.618 26.455 1739 ASP228 O −11.944 23.836 25.52 1740 ASP228 CB −13.961 26.399 26.323 1741 ASP228 CG −14.816 25.698 25.268 1742 ASP228 OD1 −14.745 24.475 25.201 1743 ASP228 OD2 −15.656 26.364 24.686 1744 HIS229 N −12.329 24.215 27.704 1745 HIS229 CA −11.958 22.854 28.129 1746 HIS229 C −12.84 21.733 27.559 1747 HIS229 O −12.334 20.622 27.37 1748 HIS229 CB −11.971 22.807 29.658 1749 HIS229 CG −13.265 23.255 30.319 1750 HIS229 ND1 −14.34 22.487 30.584 1751 HIS229 CD2 −13.557 24.519 30.78 1752 HIS229 CE1 −15.293 23.238 31.171 1753 HIS229 NE2 −14.808 24.494 31.292 1754 GLY230 N −14.024 22.066 27.068 1755 GLY230 CA −14.88 21.073 26.412 1756 GLY230 C −14.27 20.684 25.07 1757 GLY230 O −13.958 19.508 24.84 1758 GLU231 N −13.866 21.704 24.329 1759 GLU231 CA −13.239 21.508 23.021 1760 GLU231 C −11.809 21.001 23.152 1761 GLU231 O −11.365 20.21 22.315 1762 GLU231 CB −13.196 22.856 22.318 1763 GLU231 CG −14.589 23.417 22.086 1764 GLU231 CD −14.471 24.888 21.707 1765 GLU231 OE1 −15.12 25.296 20.756 1766 GLU231 OE2 −13.758 25.592 22.418 1767 LEU232 N −11.181 21.28 24.281 1768 LEU232 CA −9.822 20.803 24.524 1769 LEU232 C −9.841 19.292 24.755 1770 LEU232 O −9.061 18.578 24.112 1771 LEU232 CB −9.297 21.541 25.756 1772 LEU232 CG −7.777 21.685 25.784 1773 LEU232 CD1 −7.057 20.385 26.124 1774 LEU232 CD2 −7.256 22.293 24.486 1775 VAL233 N −10.877 18.804 25.419 1776 VAL233 CA −11.016 17.36 25.627 1777 VAL233 C −11.406 16.646 24.331 1778 VAL233 O −10.729 15.676 23.963 1779 VAL233 CB −12.066 17.13 26.71 1780 VAL233 CG1 −12.478 15.667 26.802 1781 VAL233 CG2 −11.571 17.632 28.062 1782 GLY234 N −12.258 17.278 23.536 1783 GLY234 CA −12.641 16.729 22.227 1784 GLY234 C −11.443 16.59 21.287 1785 GLY234 O −11.116 15.477 20.849 1786 LEU235 N −10.687 17.668 21.146 1787 LEU235 CA −9.532 17.691 20.238 1788 LEU235 C −8.375 16.809 20.702 1789 LEU235 O −7.846 16.046 19.882 1790 LEU235 CB −9.04 19.13 20.146 1791 LEU235 CG −10.08 20.034 19.495 1792 LEU235 CD1 −9.781 21.505 19.761 1793 LEU235 CD2 −10.2 19.75 18.003 1794 ALA236 N −8.162 16.713 22.006 1795 ALA236 CA −7.065 15.885 22.523 1796 ALA236 C −7.38 14.407 22.375 1797 ALA236 O −6.525 13.633 21.922 1798 ALA236 CB −6.861 16.193 24.002 1799 PHE237 N −8.66 14.095 22.475 1800 PHE237 CA −9.11 12.723 22.306 1801 PHE237 C −8.956 12.273 20.864 1802 PHE237 O −8.27 11.274 20.617 1803 PHE237 CB −10.58 12.657 22.682 1804 PHE237 CG −11.12 11.24 22.674 1805 PHE237 CD1 −10.824 10.394 23.733 1806 PHE237 CD2 −11.885 10.786 21.608 1807 PHE237 CE1 −11.305 9.095 23.736 1808 PHE237 CE2 −12.366 9.486 21.61 1809 PHE237 CZ −12.076 8.644 22.676 1810 LEU238 N −9.329 13.135 19.931 1811 LEU238 CA −9.272 12.754 18.516 1812 LEU238 C −7.845 12.681 17.984 1813 LEU238 O −7.532 11.745 17.236 1814 LEU238 CB −10.056 13.766 17.695 1815 LEU238 CG −11.539 13.75 18.042 1816 LEU238 CD1 −12.279 14.795 17.221 1817 LEU238 CD2 −12.145 12.369 17.814 1818 LEU239 N −6.947 13.467 18.554 1819 LEU239 CA −5.551 13.397 18.122 1820 LEU239 C −4.861 12.158 18.68 1821 LEU239 O −4.202 11.444 17.913 1822 LEU239 CB −4.821 14.652 18.586 1823 LEU239 CG −5.364 15.898 17.894 1824 LEU239 CD1 −4.722 17.162 18.454 1825 LEU239 CD2 −5.169 15.82 16.384 1826 LEU240 N −5.275 11.736 19.864 1827 LEU240 CA −4.667 10.56 20.485 1828 LEU240 C −5.217 9.265 19.877 1829 LEU240 O −4.445 8.316 19.674 1830 LEU240 CB −4.952 10.634 21.981 1831 LEU240 CG −3.966 9.808 22.798 1832 LEU240 CD1 −2.53 10.2 22.479 1833 LEU240 CD2 −4.227 9.976 24.289 1834 ILE241 N −6.425 9.337 19.333 1835 ILE241 CA −7.027 8.197 18.621 1836 ILE241 C −6.509 8.091 17.184 1837 ILE241 O −6.406 6.98 16.64 1838 ILE241 CB −8.544 8.391 18.599 1839 ILE241 CG1 −9.122 8.403 20.008 1840 ILE241 CG2 −9.233 7.311 17.771 1841 ILE241 CD1 −8.939 7.06 20.699 1842 ALA242 N −5.959 9.182 16.676 1843 ALA242 CA −5.353 9.154 15.345 1844 ALA242 C −4.049 8.363 15.361 1845 ALA242 O −3.842 7.511 14.487 1846 ALA242 CB −5.09 10.583 14.884 1847 GLY243 N −3.34 8.426 16.473 1848 GLY243 CA −2.135 7.612 16.614 1849 GLY243 C −2.461 6.175 17.012 1850 GLY243 O −2.095 5.238 16.289 1851 HIS244 N −3.274 6.021 18.047 1852 HIS244 CA −3.556 4.687 18.603 1853 HIS244 C −4.424 3.78 17.729 1854 HIS244 O −4.288 2.555 17.808 1855 HIS244 CB −4.271 4.846 19.946 1856 HIS244 CG −3.394 5.206 21.132 1857 HIS244 ND1 −2.995 6.436 21.502 1858 HIS244 CD2 −2.87 4.327 22.051 1859 HIS244 CE1 −2.232 6.348 22.61 1860 HIS244 NE2 −2.156 5.042 22.95 1861 GLU245 N −5.298 4.33 16.905 1862 GLU245 CA −6.157 3.454 16.101 1863 GLU245 C −6.041 3.723 14.61 1864 GLU245 O −5.883 2.788 13.812 1865 GLU245 CB −7.616 3.66 16.504 1866 GLU245 CG −7.895 3.313 17.963 1867 GLU245 CD −7.613 1.839 18.255 1868 GLU245 OE1 −7.721 1.028 17.348 1869 GLU245 OE2 −7.228 1.562 19.381 1870 THR246 N −5.987 4.996 14.26 1871 THR246 CA −6.101 5.374 12.849 1872 THR246 C −4.863 4.962 12.066 1873 THR246 O −4.949 4.04 11.244 1874 THR246 CB −6.314 6.88 12.759 1875 THR246 OG1 −7.415 7.225 13.59 1876 THR246 CG2 −6.614 7.344 11.338 1877 THR247 N −3.702 5.384 12.532 1878 THR247 CA −2.48 5.072 11.792 1879 THR247 C −2.013 3.634 12.044 1880 THR247 O −1.437 3.032 11.132 1881 THR247 CB −1.4 6.078 12.171 1882 THR247 OG1 −1.937 7.39 12.062 1883 THR247 CG2 −0.192 5.981 11.246 1884 ALA248 N −2.533 3.007 13.09 1885 ALA248 CA −2.225 1.596 13.35 1886 ALA248 C −2.915 0.676 12.342 1887 ALA248 O −2.236 −0.089 11.643 1888 ALA248 CB −2.678 1.25 14.763 1889 ASN249 N −4.175 0.958 12.049 1890 ASN249 CA −4.878 0.157 11.042 1891 ASN249 C −4.453 0.517 9.624 1892 ASN249 O −4.313 −0.39 8.791 1893 ASN249 CB −6.377 0.361 11.199 1894 ASN249 CG −6.902 −0.561 12.291 1895 ASN249 OD1 −6.261 −1.569 12.613 1896 ASN249 ND2 −8.133 −0.32 12.703 1897 MET250 N −3.945 1.727 9.463 1898 MET250 CA −3.472 2.183 8.159 1899 MET250 C −2.137 1.529 7.787 1900 MET250 O −2.018 1.009 6.669 1901 MET250 CB −3.231 3.702 8.255 1902 MET250 CG −3.343 4.33 6.851 1903 MET250 SD −2.234 5.768 6.665 1904 MET250 CE −1.954 6.241 8.406 1905 ILE251 N −1.267 1.317 8.764 1906 ILE251 CA 0.019 0.667 8.477 1907 ILE251 C −0.132 −0.844 8.328 1908 ILE251 O 0.449 −1.427 7.402 1909 ILE251 CB 1.001 0.949 9.613 1910 ILE251 CG1 1.293 2.434 9.757 1911 ILE251 CG2 2.305 0.188 9.404 1912 ILE251 CD1 2.264 2.698 10.902 1913 SER252 N −1.081 −1.421 9.047 1914 SER252 CA −1.254 −2.875 8.973 1915 SER252 C −1.947 −3.301 7.679 1916 SER252 O −1.444 −4.204 6.998 1917 SER252 CB −2.03 −3.36 10.197 1918 SER252 OG −3.28 −2.684 10.279 1919 LEU253 N −2.85 −2.465 7.194 1920 LEU253 CA −3.558 −2.762 5.948 1921 LEU253 C −2.699 −2.386 4.739 1922 LEU253 O −2.665 −3.129 3.748 1923 LEU253 CB −4.857 −1.963 5.994 1924 LEU253 CG −5.781 −2.185 4.804 1925 LEU253 CD1 −6.009 −3.665 4.521 1926 LEU253 CD2 −7.109 −1.479 5.058 1927 GLY254 N −1.818 −1.42 4.948 1928 GLY254 CA −0.841 −1.031 3.933 1929 GLY254 C 0.161 −2.148 3.671 1930 GLY254 O 0.267 −2.62 2.531 1931 THR255 N 0.707 −2.706 4.742 1932 THR255 CA 1.711 −3.772 4.62 1933 THR255 C 1.123 −5.042 4.017 1934 THR255 O 1.715 −5.592 3.079 1935 THR255 CB 2.255 −4.105 6.007 1936 THR255 OG1 2.837 −2.935 6.563 1937 THR255 CG2 3.334 −5.18 5.938 1938 VAL256 N −0.133 −5.321 4.331 1939 VAL256 CA −0.797 −6.505 3.781 1940 VAL256 C −1.11 −6.37 2.291 1941 VAL256 O −0.832 −7.307 1.531 1942 VAL256 CB −2.083 −6.706 4.571 1943 VAL256 CG1 −3.028 −7.693 3.905 1944 VAL256 CG2 −1.774 −7.144 5.993 1945 THR257 N −1.372 −5.158 1.833 1946 THR257 CA −1.675 −4.982 0.413 1947 THR257 C −0.401 −4.902 −0.427 1948 THR257 O −0.357 −5.499 −1.512 1949 THR257 CB −2.502 −3.717 0.248 1950 THR257 OG1 −3.63 −3.818 1.106 1951 THR257 CG2 −3.002 −3.558 −1.183 1952 LEU258 N 0.69 −4.465 0.185 1953 LEU258 CA 1.976 −4.434 −0.526 1954 LEU258 C 2.588 −5.828 −0.61 1955 LEU258 O 3.147 −6.205 −1.648 1956 LEU258 CB 2.936 −3.524 0.233 1957 LEU258 CG 2.45 −2.08 0.263 1958 LEU258 CD1 3.329 −1.229 1.172 1959 LEU258 CD2 2.391 −1.492 −1.141 1960 LEU259 N 2.248 −6.652 0.368 1961 LEU259 CA 2.721 −8.034 0.407 1962 LEU259 C 1.818 −8.965 −0.407 1963 LEU259 O 2.223 −10.083 −0.747 1964 LEU259 CB 2.742 −8.462 1.869 1965 LEU259 CG 3.979 −9.291 2.183 1966 LEU259 CD1 5.235 −8.559 1.727 1967 LEU259 CD2 4.05 −9.61 3.672 1968 GLU260 N 0.649 −8.473 −0.791 1969 GLU260 CA −0.221 −9.215 −1.707 1970 GLU260 C 0.163 −8.932 −3.151 1971 GLU260 O 0.033 −9.804 −4.019 1972 GLU260 CB −1.67 −8.773 −1.531 1973 GLU260 CG −2.53 −9.85 −0.883 1974 GLU260 CD −2.442 −9.78 0.637 1975 GLU260 OE1 −1.632 −10.498 1.204 1976 GLU260 OE2 −3.287 −9.103 1.202 1977 ASN261 N 0.693 −7.743 −3.382 1978 ASN261 CA 1.158 −7.381 −4.723 1979 ASN261 C 2.669 −7.181 −4.742 1980 ASN261 O 3.134 −6.032 −4.804 1981 ASN261 CB 0.468 −6.089 −5.161 1982 ASN261 CG −0.986 −6.311 −5.586 1983 ASN261 OD1 −1.784 −6.942 −4.881 1984 ASN261 ND2 −1.339 −5.701 −6.705 1985 PRO262 N 3.404 −8.259 −4.986 1986 PRO262 CA 4.864 −8.219 −4.846 1987 PRO262 C 5.563 −7.432 −5.958 1988 PRO262 O 6.612 −6.834 −5.706 1989 PRO262 CB 5.298 −9.652 −4.871 1990 PRO262 CG 4.108 −10.533 −5.223 1991 PRO262 CD 2.912 −9.601 −5.319 1992 ASP263 N 4.884 −7.222 −7.077 1993 ASP263 CA 5.442 −6.411 −8.166 1994 ASP263 C 5.27 −4.909 −7.918 1995 ASP263 O 6.133 −4.124 −8.327 1996 ASP263 CB 4.783 −6.822 −9.488 1997 ASP263 CG 3.253 −6.778 −9.42 1998 ASP263 OD1 2.69 −5.734 −9.721 1999 ASP263 OD2 2.673 −7.766 −8.992 2000 GLN264 N 4.358 −4.558 −7.024 2001 GLN264 CA 4.149 −3.154 −6.675 2002 GLN264 C 5.104 −2.803 −5.545 2003 GLN264 O 5.782 −1.769 −5.59 2004 GLN264 CB 2.709 −3.002 −6.206 2005 GLN264 CG 1.723 −3.483 −7.265 2006 GLN264 CD 1.637 −2.485 −8.412 2007 GLN264 OE1 1.631 −1.274 −8.178 2008 GLN264 NE2 1.592 −2.991 −9.63 2009 LEU265 N 5.398 −3.825 −4.758 2010 LEU265 CA 6.389 −3.701 −3.693 2011 LEU265 C 7.808 −3.677 −4.263 2012 LEU265 O 8.639 −2.888 −3.798 2013 LEU265 CB 6.218 −4.903 −2.775 2014 LEU265 CG 7.134 −4.809 −1.565 2015 LEU265 CD1 6.869 −3.518 −0.802 2016 LEU265 CD2 6.958 −6.023 −0.662 2017 ALA266 N 7.993 −4.309 −5.411 2018 ALA266 CA 9.286 −4.267 −6.093 2019 ALA266 C 9.528 −2.929 −6.785 2020 ALA266 O 10.66 −2.434 −6.737 2021 ALA266 CB 9.333 −5.39 −7.123 2022 LYS267 N 8.466 −2.235 −7.167 2023 LYS267 CA 8.641 −0.891 −7.725 2024 LYS267 C 8.887 0.136 −6.625 2025 LYS267 O 9.706 1.042 −6.818 2026 LYS267 CB 7.406 −0.501 −8.523 2027 LYS267 CG 7.223 −1.394 −9.742 2028 LYS267 CD 6.072 −0.894 −10.604 2029 LYS267 CE 4.779 −0.825 −9.803 2030 LYS267 NZ 3.688 −0.267 −10.615 2031 ILE268 N 8.413 −0.162 −5.427 2032 ILE268 CA 8.717 0.664 −4.254 2033 ILE268 C 10.181 0.531 −3.84 2034 ILE268 O 10.879 1.544 −3.697 2035 ILE268 CB 7.827 0.171 −3.117 2036 ILE268 CG1 6.38 0.576 −3.335 2037 ILE268 CG2 8.311 0.638 −1.75 2038 ILE268 CD1 5.504 0.057 −2.205 2039 LYS269 N 10.693 −0.688 −3.902 2040 LYS269 CA 12.068 −0.95 −3.463 2041 LYS269 C 13.116 −0.675 −4.542 2042 LYS269 O 14.306 −0.554 −4.228 2043 LYS269 CB 12.126 −2.405 −3.021 2044 LYS269 CG 11.167 −2.628 −1.858 2045 LYS269 CD 10.997 −4.107 −1.542 2046 LYS269 CE 12.315 −4.756 −1.148 2047 LYS269 NZ 12.106 −6.181 −0.856 2048 ALA270 N 12.679 −0.53 −5.782 2049 ALA270 CA 13.585 −0.106 −6.851 2050 ALA270 C 13.478 1.396 −7.101 2051 ALA270 O 14.286 1.974 −7.838 2052 ALA270 CB 13.233 −0.863 −8.125 2053 ASP271 N 12.486 2.017 −6.486 2054 ASP271 CA 12.271 3.453 −6.649 2055 ASP271 C 11.505 4.006 −5.448 2056 ASP271 O 10.267 4.02 −5.445 2057 ASP271 CB 11.48 3.653 −7.944 2058 ASP271 CG 11.354 5.125 −8.337 2059 ASP271 OD1 10.975 5.919 −7.482 2060 ASP271 OD2 11.493 5.405 −9.517 2061 PRO272 N 12.238 4.705 −4.592 2062 PRO272 CA 11.686 5.248 −3.337 2063 PRO272 C 10.72 6.438 −3.497 2064 PRO272 O 9.973 6.74 −2.558 2065 PRO272 CB 12.887 5.657 −2.54 2066 PRO272 CG 14.133 5.544 −3.406 2067 PRO272 CD 13.673 4.968 −4.734 2068 GLY273 N 10.586 6.971 −4.702 2069 GLY273 CA 9.607 8.03 −4.968 2070 GLY273 C 8.207 7.423 −5.037 2071 GLY273 O 7.257 7.968 −4.456 2072 LYS274 N 8.167 6.171 −5.473 2073 LYS274 CA 6.916 5.418 −5.566 2074 LYS274 C 6.39 4.954 −4.21 2075 LYS274 O 5.225 4.55 −4.144 2076 LYS274 CB 7.138 4.184 −6.431 2077 LYS274 CG 7.528 4.547 −7.856 2078 LYS274 CD 7.755 3.287 −8.681 2079 LYS274 CE 8.177 3.615 −10.107 2080 LYS274 NZ 8.465 2.383 −10.858 2081 THR275 N 7.12 5.182 −3.127 2082 THR275 CA 6.593 4.814 −1.813 2083 THR275 C 5.522 5.804 −1.352 2084 THR275 O 4.516 5.354 −0.798 2085 THR275 CB 7.725 4.76 −0.789 2086 THR275 OG1 8.169 6.074 −0.485 2087 THR275 CG2 8.912 3.963 −1.305 2088 LEU276 N 5.564 7.036 −1.844 2089 LEU276 CA 4.543 8.012 −1.446 2090 LEU276 C 3.312 7.905 −2.346 2091 LEU276 O 2.175 8.051 −1.876 2092 LEU276 CB 5.14 9.411 −1.538 2093 LEU276 CG 4.182 10.462 −0.987 2094 LEU276 CD1 3.836 10.177 0.472 2095 LEU276 CD2 4.77 11.861 −1.133 2096 ALA277 N 3.53 7.375 −3.539 2097 ALA277 CA 2.417 7.126 −4.451 2098 ALA277 C 1.711 5.836 −4.052 2099 ALA277 O 0.475 5.796 −4.026 2100 ALA277 CB 2.963 7.021 −5.869 2101 ALA278 N 2.472 4.947 −3.431 2102 ALA278 CA 1.909 3.727 −2.859 2103 ALA278 C 1.12 4.01 −1.591 2104 ALA278 O 0.051 3.416 −1.427 2105 ALA278 CB 3.048 2.773 −2.523 2106 ILE279 N 1.472 5.064 −0.867 2107 ILE279 CA 0.698 5.447 0.324 2108 ILE279 C −0.692 5.926 −0.078 2109 ILE279 O −1.691 5.368 0.396 2110 ILE279 CB 1.401 6.588 1.063 2111 ILE279 CG1 2.806 6.209 1.513 2112 ILE279 CG2 0.577 7.041 2.264 2113 ILE279 CD1 2.805 4.984 2.416 2114 GLU280 N −0.748 6.688 −1.159 2115 GLU280 CA −2.037 7.217 −1.616 2116 GLU280 C −2.849 6.159 −2.358 2117 GLU280 O −4.075 6.123 −2.217 2118 GLU280 CB −1.784 8.404 −2.537 2119 GLU280 CG −0.943 9.484 −1.862 2120 GLU280 CD −1.616 9.992 −0.587 2121 GLU280 OE1 −2.453 10.875 −0.699 2122 GLU280 OE2 −1.19 9.569 0.479 2123 GLU281 N −2.169 5.173 −2.916 2124 GLU281 CA −2.857 4.083 −3.604 2125 GLU281 C −3.402 3.048 −2.616 2126 GLU281 O −4.497 2.514 −2.842 2127 GLU281 CB −1.846 3.441 −4.546 2128 GLU281 CG −2.451 2.329 −5.39 2129 GLU281 CD −3.395 2.862 −6.465 2130 GLU281 OE1 −3.893 2.017 −7.199 2131 GLU281 OE2 −3.446 4.068 −6.653 2132 LEU282 N −2.776 2.957 −1.452 2133 LEU282 CA −3.272 2.102 −0.369 2134 LEU282 C −4.527 2.703 0.232 2135 LEU282 O −5.563 2.03 0.269 2136 LEU282 CB −2.212 2.019 0.727 2137 LEU282 CG −1.025 1.151 0.332 2138 LEU282 CD1 0.163 1.385 1.256 2139 LEU282 CD2 −1.415 −0.319 0.305 2140 LEU283 N −4.511 4.018 0.378 2141 LEU283 CA −5.666 4.738 0.922 2142 LEU283 C −6.823 4.808 −0.066 2143 LEU283 O −7.989 4.766 0.346 2144 LEU283 CB −5.202 6.145 1.257 2145 LEU283 CG −4.178 6.103 2.379 2146 LEU283 CD1 −3.386 7.399 2.461 2147 LEU283 CD2 −4.848 5.766 3.706 2148 ARG284 N −6.512 4.723 −1.346 2149 ARG284 CA −7.561 4.67 −2.355 2150 ARG284 C −8.306 3.337 −2.3 2151 ARG284 O −9.482 3.316 −1.923 2152 ARG284 CB −6.921 4.824 −3.726 2153 ARG284 CG −7.99 4.956 −4.798 2154 ARG284 CD −7.417 4.674 −6.18 2155 ARG284 NE −6.879 3.305 −6.258 2156 ARG284 CZ −7.603 2.231 −6.583 2157 ARG284 NH1 −8.911 2.35 −6.816 2158 ARG284 NH2 −7.021 1.032 −6.658 2159 ILE285 N −7.588 2.233 −2.423 2160 ILE285 CA −8.284 0.942 −2.519 2161 ILE285 C −8.742 0.397 −1.155 2162 ILE285 O −9.703 −0.385 −1.087 2163 ILE285 CB −7.36 −0.039 −3.247 2164 ILE285 CG1 −8.033 −1.383 −3.499 2165 ILE285 CG2 −6.045 −0.238 −2.501 2166 ILE285 CD1 −7.119 −2.327 −4.272 2167 PHE286 N −8.188 0.934 −0.082 2168 PHE286 CA −8.543 0.5 1.268 2169 PHE286 C −8.451 1.645 2.27 2170 PHE286 O −7.544 1.66 3.115 2171 PHE286 CB −7.575 −0.594 1.708 2172 PHE286 CG −7.737 −1.948 1.025 2173 PHE286 CD1 −6.675 −2.503 0.323 2174 PHE286 CD2 −8.943 −2.632 1.117 2175 PHE286 CE1 −6.822 −3.736 −0.298 2176 PHE286 CE2 −9.09 −3.865 0.495 2177 PHE286 CZ −8.03 −4.417 −0.213 2178 THR287 N −9.377 2.584 2.193 2179 THR287 CA −9.413 3.636 3.212 2180 THR287 C −9.931 3.053 4.519 2181 THR287 O −10.843 2.216 4.543 2182 THR287 CB −10.294 4.801 2.77 2183 THR287 OG1 −10.207 5.833 3.745 2184 THR287 CG2 −11.759 4.423 2.643 2185 ILE288 N −9.305 3.459 5.609 2186 ILE288 CA −9.745 2.966 6.911 2187 ILE288 C −10.966 3.747 7.391 2188 ILE288 O −11.856 3.155 8.014 2189 ILE288 CB −8.584 3.044 7.894 2190 ILE288 CG1 −8.04 4.459 8.013 2191 ILE288 CG2 −7.469 2.097 7.461 2192 ILE288 CD1 −6.938 4.507 9.052 2193 ALA289 N −11.116 4.965 6.89 2194 ALA289 CA −12.34 5.743 7.09 2195 ALA289 C −13.315 5.37 5.982 2196 ALA289 O −13.433 6.062 4.964 2197 ALA289 CB −12.003 7.228 7.013 2198 GLU290 N −13.969 4.239 6.174 2199 GLU290 CA −14.796 3.652 5.128 2200 GLU290 C −16.223 4.179 5.178 2201 GLU290 O −16.891 4.234 4.138 2202 GLU290 CB −14.766 2.14 5.34 2203 GLU290 CG −15.669 1.375 4.381 2204 GLU290 CD −15.736 −0.091 4.802 2205 GLU290 OE1 −16.751 −0.475 5.367 2206 GLU290 OE2 −14.809 −0.819 4.472 2207 THR291 N −16.659 4.619 6.347 2208 THR291 CA −17.992 5.216 6.481 2209 THR291 C −17.983 6.47 7.349 2210 THR291 O −17.68 6.437 8.55 2211 THR291 CB −18.978 4.219 7.094 2212 THR291 OG1 −18.522 3.832 8.382 2213 THR291 CG2 −19.168 2.961 6.257 2214 ALA292 N −18.397 7.562 6.734 2215 ALA292 CA −18.676 8.799 7.471 2216 ALA292 C −20.103 8.704 8.001 2217 ALA292 O −21.051 9.169 7.355 2218 ALA292 CB −18.552 9.986 6.521 2219 THR293 N −20.231 8.1 9.172 2220 THR293 CA −21.535 7.709 9.721 2221 THR293 C −22.161 8.802 10.585 2222 THR293 O −22.152 8.733 11.82 2223 THR293 CB −21.308 6.447 10.546 2224 THR293 OG1 −20.564 5.523 9.756 2225 THR293 CG2 −22.617 5.788 10.962 2226 SER294 N −22.703 9.805 9.913 2227 SER294 CA −23.293 10.956 10.601 2228 SER294 C −24.198 11.766 9.675 2229 SER294 O −25.386 11.443 9.529 2230 SER294 CB −22.167 11.822 11.164 2231 SER294 OG −20.976 11.559 10.426 2232 ARG295 N −23.598 12.789 9.076 2233 ARG295 CA −24.233 13.782 8.187 2234 ARG295 C −25.738 13.903 8.366 2235 ARG295 O −26.524 13.3 7.627 2236 ARG295 CB −23.905 13.424 6.747 2237 ARG295 CG −22.4 13.455 6.518 2238 ARG295 CD −21.819 14.841 6.786 2239 ARG295 NE −20.362 14.859 6.577 2240 ARG295 CZ −19.475 14.679 7.56 2241 ARG295 NH1 −19.892 14.437 8.805 2242 ARG295 NH2 −18.167 14.719 7.293 2243 PHE296 N −26.119 14.646 9.387 2244 PHE296 GA −27.528 14.825 9.712 2245 PHE296 C −28.176 15.754 8.701 2246 PHE296 O −27.736 16.899 8.529 2247 PHE296 CB −27.623 15.428 11.11 2248 PHE296 CG −29.052 15.695 11.562 2249 PHE296 CD1 −29.564 16.985 11.533 2250 PHE296 CD2 −29.843 14.643 12.002 2251 PHE296 CE1 −30.871 17.221 11.936 2252 PHE296 CE2 −31.149 14.879 12.406 2253 PHE296 CZ −31.663 16.167 12.37 2254 ALA297 N −29.2 15.255 8.032 2255 ALA297 CA −29.931 16.073 7.065 2256 ALA297 C −30.783 17.111 7.781 2257 ALA297 O −31.769 16.781 8.45 2258 ALA297 CB −30.819 15.165 6.224 2259 THR298 N −30.369 18.361 7.668 2260 THR298 CA −31.129 19.47 8.255 2261 THR298 C −32.139 20.005 7.246 2262 THR298 O −33.087 20.712 7.603 2263 THR298 CB −30.17 20.586 8.659 2264 THR298 OG1 −29.565 21.12 7.488 2265 THR298 CG2 −29.068 20.082 9.582 2266 ALA299 N −31.926 19.649 5.992 2267 ALA299 CA −32.881 19.968 4.932 2268 ALA299 C −33.091 18.734 4.068 2269 ALA299 O −32.427 17.712 4.27 2270 ALA299 CB −32.331 21.11 4.086 2271 ASP300 N −34.041 18.817 3.151 2272 ASP300 CA −34.256 17.727 2.191 2273 ASP300 C −33.253 17.852 1.048 2274 ASP300 O −33.498 18.545 0.053 2275 ASP300 CB −35.679 17.801 1.648 2276 ASP300 CG −36.682 17.714 2.793 2277 ASP300 OD1 −36.686 16.69 3.463 2278 ASP300 OD2 −37.289 18.733 3.09 2279 VAL301 N −32.128 17.177 1.199 2280 VAL301 CA −31.029 17.337 0.244 2281 VAL301 C −31.065 16.261 −0.833 2282 VAL301 O −30.956 15.062 −0.55 2283 VAL301 CB −29.711 17.263 1.007 2284 VAL301 CG1 −28.543 17.673 0.118 2285 VAL301 CG2 −29.756 18.154 2.24 2286 GLU302 N −31.242 16.695 −2.067 2287 GLU302 CA −31.23 15.75 −3.184 2288 GLU302 C −29.799 15.413 −3.603 2289 GLU302 O −29.141 16.178 −4.318 2290 GLU302 CB −31.98 16.355 −4.363 2291 GLU302 CG −32.053 15.344 −5.497 2292 GLU302 CD −32.662 15.959 −6.75 2293 GLU302 OE1 −33.484 16.852 −6.606 2294 GLU302 OE2 −32.169 15.628 −7.82 2295 ILE303 N −29.365 14.227 −3.222 2296 ILE303 CA −28.014 13.766 −3.539 2297 ILE303 C −28.053 12.812 −4.726 2298 ILE303 O −28.452 11.647 −4.6 2299 ILE303 CB −27.438 13.076 −2.307 2300 ILE303 CG1 −27.329 14.056 −1.148 2301 ILE303 CG2 −26.066 12.49 −2.608 2302 ILE303 CD1 −26.305 15.146 −1.441 2303 GLY304 N −27.715 13.356 −5.885 2304 GLY304 CA −27.705 12.592 −7.139 2305 GLY304 C −29.056 11.935 −7.402 2306 GLY304 O −29.187 10.708 −7.311 2307 GLY305 N −30.079 12.757 −7.573 2308 GLY305 CA −31.432 12.229 −7.805 2309 GLY305 C −32.226 11.982 −6.517 2310 GLY305 O −33.289 12.584 −6.314 2311 THR306 N −31.694 11.132 −5.653 2312 THR306 CA −32.399 10.717 −4.431 2313 THR306 C −32.569 11.859 −3.43 2314 THR306 O −31.597 12.497 −3.015 2315 THR306 CB −31.591 9.598 −3.782 2316 THR306 OG1 −31.461 8.534 −4.716 2317 THR306 CG2 −32.278 9.051 −2.537 2318 LEU307 N −33.811 12.127 −3.066 2319 LEU307 CA −34.093 13.162 −2.068 2320 LEU307 C −33.971 12.6 −0.652 2321 LEU307 O −34.803 11.799 −0.209 2322 LEU307 CB −35.513 13.67 −2.295 2323 LEU307 CG −35.845 14.852 −1.391 2324 LEU307 CD1 −34.946 16.039 −1.71 2325 LEU307 CD2 −37.31 15.248 −1.534 2326 ILE308 N −32.93 13.021 0.045 2327 ILE308 CA −32.738 12.616 1.439 2328 ILE308 C −33.586 13.486 2.362 2329 ILE308 O −33.481 14.719 2.356 2330 ILE308 CB −31.253 12.742 1.764 2331 ILE308 CG1 −30.456 11.77 0.901 2332 ILE308 CG2 −30.976 12.507 3.245 2333 ILE308 CD1 −28.974 11.794 1.246 2334 ARG309 N −34.466 12.834 3.101 2335 ARG309 CA −35.386 13.543 3.993 2336 ARG309 C −34.674 14.125 5.209 2337 ARG309 O −33.807 13.486 5.824 2338 ARG309 CB −36.447 12.563 4.474 2339 ARG309 CG −37.117 11.841 3.312 2340 ARG309 CD −38.079 10.778 3.83 2341 ARG309 NE −38.721 10.055 2.721 2342 ARG309 CZ −40.046 9.951 2.594 2343 ARG309 NH1 −40.85 10.528 3.49 2344 ARG309 NH2 −40.566 9.278 1.566 2345 ALA310 N −35.099 15.325 5.566 2346 ALA310 CA −34.597 15.999 6.763 2347 ALA310 C −34.952 15.189 8.003 2348 ALA310 O −36.005 14.542 8.07 2349 ALA310 CB −35.223 17.386 6.853 2350 GLY311 N −34 15.115 8.913 2351 GLY311 CA −34.172 14.325 10.13 2352 GLY311 C −33.475 12.968 10.042 2353 GLY311 O −33.472 12.217 11.023 2354 GLU312 N −32.982 12.611 8.866 2355 GLU312 CA −32.294 11.322 8.719 2356 GLU312 C −30.776 11.471 8.737 2357 GLU312 O −30.236 12.577 8.599 2358 GLU312 CB −32.743 10.651 7.429 2359 GLU312 CG −34.243 10.386 7.453 2360 GLU312 CD −34.639 9.537 6.252 2361 GLU312 OE1 −34.686 10.083 5.156 2362 GLU312 OE2 −34.723 8.33 6.419 2363 GLY313 N −30.107 10.356 8.981 2364 GLY313 CA −28.64 10.335 8.994 2365 GLY313 C −28.079 9.813 7.673 2366 GLY313 O −28.674 8.944 7.02 2367 VAL314 N −26.996 10.429 7.237 2368 VAL314 CA −26.33 10.028 5.994 2369 VAL314 C −24.93 9.467 6.267 2370 VAL314 O −24.127 10.02 7.033 2371 VAL314 CB −26.28 11.246 5.073 2372 VAL314 CG1 −25.639 10.942 3.724 2373 VAL314 CG2 −27.681 11.804 4.861 2374 VAL315 N −24.674 8.328 5.649 2375 VAL315 CA −23.386 7.646 5.773 2376 VAL315 C −22.647 7.666 4.435 2377 VAL315 O −23.025 6.975 3.479 2378 VAL315 CB −23.634 6.207 6.213 2379 VAL315 CG1 −22.325 5.437 6.347 2380 VAL315 CG2 −24.404 6.163 7.528 2381 GLY316 N −21.6 8.467 4.374 2382 GLY316 CA −20.789 8.539 3.152 2383 GLY316 C −19.797 7.382 3.096 2384 GLY316 O −18.903 7.28 3.946 2385 LEU317 N −19.978 6.508 2.121 2386 LEU317 CA −19.102 5.343 1.978 2387 LEU317 C −17.841 5.722 1.218 2388 LEU317 O −17.758 5.485 0.009 2389 LEU317 CB −19.835 4.256 1.199 2390 LEU317 CG −20.312 3.094 2.064 2391 LEU317 CD1 −19.129 2.374 2.697 2392 LEU317 CD2 −21.328 3.519 3.121 2393 SER318 N −16.794 6.056 1.951 2394 SER318 CA −15.563 6.559 1.34 2395 SER318 C −14.789 5.483 0.588 2396 SER318 O −14.286 5.777 −0.503 2397 SER318 CB −14.689 7.158 2.434 2398 SER318 OG −13.375 7.32 1.915 2399 ASN319 N −14.954 4.222 0.954 2400 ASN319 CA −14.267 3.196 0.162 2401 ASN319 C −15.089 2.774 −1.058 2402 ASN319 O −14.498 2.343 −2.052 2403 ASN319 CB −13.899 1.982 1.004 2404 ASN319 CG −12.531 1.507 0.516 2405 ASN319 OD1 −11.628 2.333 0.321 2406 ASN319 ND2 −12.378 0.209 0.332 2407 ALA320 N −16.362 3.139 −1.091 2408 ALA320 CA −17.181 2.889 −2.283 2409 ALA320 C −17.001 4.055 −3.248 2410 ALA320 O −16.897 3.856 −4.464 2411 ALA320 CB −18.642 2.78 −1.875 2412 GLY321 N −16.68 5.197 −2.666 2413 GLY321 CA −16.233 6.36 −3.426 2414 GLY321 C −14.942 6.045 −4.17 2415 GLY321 O −14.924 6.106 −5.404 2416 ASN322 N −13.958 5.508 −3.466 2417 ASN322 CA −12.683 5.172 −4.113 2418 ASN322 C −12.765 3.924 −5.006 2419 ASN322 O −11.954 3.769 −5.927 2420 ASN322 CB −11.651 4.902 −3.035 2421 ASN322 CG −11.54 6.013 −1.994 2422 ASN322 OD1 −11.701 7.211 −2.278 2423 ASN322 ND2 −11.095 5.595 −0.824 2424 HIS323 N −13.785 3.103 −4.807 2425 HIS323 CA −14.068 1.985 −5.716 2426 HIS323 C −15.037 2.337 −6.846 2427 HIS323 O −15.541 1.435 −7.525 2428 HIS323 CB −14.625 0.804 −4.939 2429 HIS323 CG −13.581 0.008 −4.19 2430 HIS323 ND1 −13.802 −0.807 −3.144 2431 HIS323 CD2 −12.233 −0.04 −4.46 2432 HIS323 CE1 −12.634 −1.351 −2.748 2433 HIS323 NE2 −11.664 −0.875 −3.563 2434 ASP324 N −15.353 3.608 −7.007 2435 ASP324 CA −16.209 4.041 −8.109 2436 ASP324 C −15.409 4.076 −9.411 2437 ASP324 O −14.628 5.012 −9.642 2438 ASP324 CB −16.72 5.431 −7.737 2439 ASP324 CG −17.711 6.002 −8.74 2440 ASP324 OD1 −18.738 6.476 −8.275 2441 ASP324 OD2 −17.28 6.257 −9.859 2442 PRO325 N −15.831 3.237 −10.349 2443 PRO325 CA −15.082 2.989 −11.595 2444 PRO325 C −15.201 4.099 −12.648 2445 PRO325 O −14.504 4.065 −13.667 2446 PRO325 CB −15.653 1.713 −12.134 2447 PRO325 CG −16.9 1.346 −11.345 2448 PRO325 CD −17.01 2.374 −10.232 2449 ASP326 N −16.008 5.11 −12.361 2450 ASP326 CA −16.206 6.248 −13.259 2451 ASP326 C −15.146 7.312 −12.983 2452 ASP326 O −14.924 8.216 −13.797 2453 ASP326 CB −17.597 6.826 −12.994 2454 ASP326 CG −18.675 5.753 −13.155 2455 ASP326 OD1 −19.125 5.571 −14.277 2456 ASP326 OD2 −18.963 5.068 −12.179 2457 GLY327 N −14.468 7.162 −11.857 2458 GLY327 CA −13.318 8.005 −11.542 2459 GLY327 C −12.061 7.145 −11.594 2460 GLY327 O −11.166 7.366 −12.418 2461 PHE328 N −12.046 6.12 −10.76 2462 PHE328 CA −10.893 5.218 −10.705 2463 PHE328 C −11.119 4.009 −11.602 2464 PHE328 O −11.92 3.114 −11.3 2465 PHE328 CB −10.653 4.81 −9.257 2466 PHE328 CG −10.261 5.995 −8.376 2467 PHE328 CD1 −9.098 6.702 −8.651 2468 PHE328 CD2 −11.07 6.379 −7.314 2469 PHE328 CE1 −8.737 7.784 −7.858 2470 PHE328 CE2 −10.709 7.459 −6.519 2471 PHE328 CZ −9.542 8.161 −6.791 2472 GLU329 N −10.377 4.006 −12.696 2473 GLU329 CA −10.511 2.993 −13.752 2474 GLU329 C −10.048 1.63 −13.253 2475 GLU329 O −8.847 1.399 −13.092 2476 GLU329 CB −9.673 3.409 −14.962 2477 GLU329 CG −10.104 4.752 −15.557 2478 GLU329 CD −9.106 5.864 −15.215 2479 GLU329 OE1 −8.54 5.804 −14.132 2480 GLU329 OE2 −8.904 6.724 −16.059 2481 ASN330 N −10.987 0.698 −13.249 2482 ASN330 CA −10.826 −0.597 −12.566 2483 ASN330 C −10.252 −0.375 −11.171 2484 ASN330 O −9.065 −0.629 −10.929 2485 ASN330 CB −9.925 −1.547 −13.355 2486 ASN330 CG −9.943 −2.959 −12.75 2487 ASN330 OD1 −9.958 −3.151 −11.523 2488 ASN330 ND2 −9.893 −3.939 −13.632 2489 PRO331 N −11.157 −0.201 −10.224 2490 PRO331 CA −10.787 0.129 −8.843 2491 PRO331 C −10.287 −1.061 −8.012 2492 PRO331 O −9.994 −0.904 −6.822 2493 PRO331 CB −12.044 0.673 −8.25 2494 PRO331 CG −13.21 0.356 −9.172 2495 PRO331 CD −12.608 −0.26 −10.416 2496 ASP332 N −10.198 −2.236 −8.615 2497 ASP332 CA −9.742 −3.417 −7.889 2498 ASP332 C −8.267 −3.667 −8.179 2499 ASP332 O −7.609 −4.447 −7.478 2500 ASP332 CB −10.557 −4.617 −8.358 2501 ASP332 CG −12.048 −4.356 −8.173 2502 ASP332 OD1 −12.43 −3.961 −7.08 2503 ASP332 OD2 −12.784 −4.566 −9.127 2504 THR333 N −7.744 −2.97 −9.173 2505 THR333 CA −6.339 −3.153 −9.535 2506 THR333 C −5.433 −2.224 −8.735 2507 THR333 O −5.283 −1.042 −9.06 2508 THR333 CB −6.189 −2.877 −11.026 2509 THR333 OG1 −7.072 −3.748 −11.718 2510 THR333 CG2 −4.769 −3.147 −11.514 2511 PHE334 N −4.84 −2.772 −7.688 2512 PHE334 CA −3.868 −2.017 −6.89 2513 PHE334 C −2.589 −1.762 −7.684 2514 PHE334 O −1.817 −2.686 −7.979 2515 PHE334 CB −3.543 −2.812 −5.63 2516 PHE334 CG −2.485 −2.162 −4.744 2517 PHE334 CD1 −2.784 −1 −4.047 2518 PHE334 CD2 −1.221 −2.728 −4.638 2519 PHE334 CE1 −1.82 −0.404 −3.246 2520 PHE334 CE2 −0.256 −2.132 −3.837 2521 PHE334 CZ −0.556 −0.969 −3.142 2522 ASP335 N −2.407 −0.509 −8.064 2523 ASP335 CA −1.206 −0.106 −8.794 2524 ASP335 C −0.697 1.24 −8.283 2525 ASP335 O −1.322 2.277 −8.525 2526 ASP335 CB −1.55 −0.019 −10.277 2527 ASP335 CG −0.276 0.2 −11.084 2528 ASP335 OD1 0.34 −0.792 −11.448 2529 ASP335 OD2 0.15 1.347 −11.159 2530 ILE336 N 0.532 1.249 −7.794 2531 ILE336 CA 1.118 2.426 −7.127 2532 ILE336 C 1.596 3.552 −8.058 2533 ILE336 O 2.207 4.512 −7.579 2534 ILE336 CB 2.303 1.948 −6.301 2535 ILE336 CG1 3.437 1.48 −7.203 2536 ILE336 CG2 1.873 0.817 −5.375 2537 ILE336 CD1 4.676 1.131 −6.396 2538 GLU337 N 1.41 3.407 −9.361 2539 GLU337 CA 1.712 4.496 −10.291 2540 GLU337 C 0.434 5.236 −10.681 2541 GLU337 O 0.487 6.246 −11.393 2542 GLU337 CB 2.393 3.936 −11.533 2543 GLU337 CG 3.788 3.412 −11.213 2544 GLU337 CD 4.442 2.871 −12.481 2545 GLU337 OE1 3.934 3.167 −13.552 2546 GLU337 OE2 5.372 2.089 −12.348 2547 ARG338 N −0.697 4.719 −10.229 2548 ARG338 CA −1.982 5.376 −10.464 2549 ARG338 C −2.095 6.636 −9.615 2550 ARG338 O −1.809 6.625 −8.412 2551 ARG338 CB −3.085 4.393 −10.082 2552 ARG338 CG −4.484 4.977 −10.234 2553 ARG338 CD −5.532 3.961 −9.809 2554 ARG338 NE −5.375 2.729 −10.591 2555 ARG338 CZ −6.357 1.847 −10.77 2556 ARG338 NH1 −7.549 2.056 −10.207 2557 ARG338 NH2 −6.143 0.757 −11.509 2558 GLY339 N −2.423 7.735 −10.271 2559 GLY339 CA −2.685 8.98 −9.55 2560 GLY339 C −4.038 8.895 −8.852 2561 GLY339 O −5.086 9.045 −9.489 2562 ALA340 N −3.994 8.767 −7.534 2563 ALA340 CA −5.202 8.646 −6.691 2564 ALA340 C −5.871 9.982 −6.335 2565 ALA340 O −6.419 10.145 −5.237 2566 ALA340 CB −4.817 7.907 −5.414 2567 ARG341 N −5.844 10.917 −7.27 2568 ARG341 CA −6.395 12.25 −7.043 2569 ARG341 C −7.904 12.179 −6.87 2570 ARG341 O −8.571 11.307 −7.436 2571 ARG341 CB −6.045 13.131 −8.235 2572 ARG341 CG −4.535 13.264 −8.387 2573 ARG341 CD −4.175 14.135 −9.583 2574 ARG341 NE −2.718 14.303 −9.691 2575 ARG341 CZ −2.142 15.463 −10.016 2576 ARG341 NH1 −2.898 16.534 −10.271 2577 ARG341 NH2 −0.813 15.551 −10.092 2578 HIS342 N −8.378 13.009 −5.954 2579 HIS342 CA −9.801 13.12 −5.603 2580 HIS342 C −10.342 11.888 −4.88 2581 HIS342 O −11.547 11.621 −4.961 2582 HIS342 CB −10.644 13.367 −6.855 2583 HIS342 CG −10.32 14.641 −7.606 2584 HIS342 ND1 −10.757 15.877 −7.303 2585 HIS342 CD2 −9.536 14.758 −8.731 2586 HIS342 CE1 −10.259 16.758 −8.195 2587 HIS342 NE2 −9.504 16.064 −9.078 2588 HIS343 N −9.49 11.15 −4.184 2589 HIS343 CA −10.02 10.075 −3.346 2590 HIS343 C −10.572 10.661 −2.053 2591 HIS343 O −9.984 11.572 −1.455 2592 HIS343 CB −8.988 8.965 −3.085 2593 HIS343 CG −7.691 9.253 −2.333 2594 HIS343 ND1 −7.374 10.313 −1.562 2595 HIS343 CD2 −6.597 8.421 −2.319 2596 HIS343 CE1 −6.125 10.166 −1.079 2597 HIS343 NE2 −5.643 8.993 −1.549 2598 VAL344 N −11.634 10.052 −1.563 2599 VAL344 CA −12.278 10.521 −0.331 2600 VAL344 C −11.732 9.82 0.915 2601 VAL344 O −12.271 9.998 2.015 2602 VAL344 CB −13.787 10.343 −0.45 2603 VAL344 CG1 −14.412 11.457 −1.282 2604 VAL344 CG2 −14.141 8.977 −1.019 2605 ALA345 N −10.581 9.178 0.768 2606 ALA345 CA −9.947 8.411 1.849 2607 ALA345 C −9.419 9.24 3.02 2608 ALA345 O −9.242 8.703 4.12 2609 ALA345 CB −8.757 7.686 1.237 2610 PHE346 N −9.169 10.518 2.787 2611 PHE346 CA −8.763 11.424 3.867 2612 PHE346 C −9.885 12.363 4.291 2613 PHE346 O −9.652 13.29 5.083 2614 PHE346 CB −7.571 12.253 3.409 2615 PHE346 CG −6.223 11.583 3.635 2616 PHE346 CD1 −5.295 11.513 2.605 2617 PHE346 CD2 −5.921 11.048 4.881 2618 PHE346 CE1 −4.061 10.916 2.825 2619 PHE346 CE2 −4.687 10.451 5.101 2620 PHE346 CZ −3.756 10.388 4.073 2621 GLY347 N −11.068 12.169 3.731 2622 GLY347 CA −12.177 13.091 3.979 2623 GLY347 C −12.007 14.367 3.158 2624 GLY347 O −10.882 14.764 2.825 2625 PHE348 N −13.123 14.988 2.822 2626 PHE348 CA −13.084 16.264 2.097 2627 PHE348 C −13.98 17.307 2.752 2628 PHE348 O −14.185 17.307 3.976 2629 PHE348 CB −13.497 16.085 0.64 2630 PHE348 CG −12.375 15.613 −0.285 2631 PHE348 CD1 −12.654 14.749 −1.333 2632 PHE348 CD2 −11.075 16.06 −0.081 2633 PHE348 CE1 −11.633 14.322 −2.172 2634 PHE348 CE2 −10.054 15.633 −0.918 2635 PHE348 CZ −10.332 14.763 −1.963 2636 GLY349 N −14.438 18.227 1.918 2637 GLY349 CA −15.269 19.339 2.376 2638 GLY349 C −14.484 20.194 3.353 2639 GLY349 O −13.286 20.433 3.165 2640 VAL350 N −15.121 20.525 4.458 2641 VAL350 CA −14.426 21.29 5.493 2642 VAL350 C −13.865 20.408 6.613 2643 VAL350 O −12.964 20.854 7.327 2644 VAL350 CB −15.384 22.334 6.062 2645 VAL350 CG1 −15.675 23.433 5.047 2646 VAL350 CG2 −16.682 21.709 6.561 2647 HIS351 N −14.217 19.133 6.631 2648 HIS351 CA −13.875 18.287 7.784 2649 HIS351 C −12.75 17.284 7.532 2650 HIS351 O −12.711 16.259 8.223 2651 HIS351 CB −15.113 17.514 8.23 2652 HIS351 CG −16.205 18.349 8.87 2653 HIS351 ND1 −16.137 18.987 10.054 2654 HIS351 CD2 −17.46 18.588 8.36 2655 HIS351 CE1 −17.304 19.619 10.29 2656 HIS351 NE2 −18.122 19.372 9.242 2657 GLN352 N −11.876 17.53 6.568 2658 GLN352 CA −10.852 16.518 6.26 2659 GLN352 C −9.793 16.385 7.357 2660 GLN352 O −9.716 17.193 8.291 2661 GLN352 CB −10.178 16.785 4.923 2662 GLN352 CG −9.244 17.98 4.902 2663 GLN352 CD −8.308 17.803 3.712 2664 GLN352 OE1 −7.316 18.528 3.568 2665 GLN352 NE2 −8.572 16.766 2.934 2666 CYS353 N −9.049 15.296 7.271 2667 CYS353 CA −8.044 14.964 8.287 2668 CYS353 C −6.883 15.962 8.319 2669 CYS353 O −6.101 16.072 7.366 2670 CYS353 CB −7.524 13.567 7.961 2671 CYS353 SG −6.412 12.816 9.17 2672 LEU354 N −6.724 16.61 9.464 2673 LEU354 CA −5.607 17.549 9.666 2674 LEU354 C −4.308 16.852 10.059 2675 LEU354 O −3.224 17.358 9.747 2676 LEU354 CB −5.967 18.561 10.748 2677 LEU354 CG −6.805 19.7 10.188 2678 LEU354 CD1 −7.148 20.701 11.284 2679 LEU354 CD2 −6.054 20.396 9.057 2680 GLY355 N −4.414 15.6 10.478 2681 GLY355 CA −3.223 14.791 10.772 2682 GLY355 C −2.835 13.936 9.566 2683 GLY355 O −2.119 12.936 9.702 2684 GLN356 N −3.078 14.499 8.393 2685 GLN356 CA −2.896 13.81 7.119 2686 GLN356 C −1.414 13.677 6.799 2687 GLN356 O −0.939 12.562 6.544 2688 GLN356 CB −3.598 14.696 6.094 2689 GLN356 CG −3.657 14.117 4.691 2690 GLN356 CD −4.524 15.024 3.818 2691 GLN356 OE1 −4.609 14.834 2.599 2692 GLN356 NE2 −5.231 15.939 4.463 2693 ASN357 N −0.67 14.7 7.191 2694 ASN357 CA 0.781 14.7 7.005 2695 ASN357 C 1.476 13.761 7.989 2696 ASN357 O 2.328 12.977 7.554 2697 ASN357 CB 1.258 16.137 7.203 2698 ASN357 CG 2.78 16.244 7.261 2699 ASN357 OD1 3.324 16.808 8.217 2700 ASN357 ND2 3.444 15.745 6.233 2701 LEU358 N 0.896 13.592 9.166 2702 LEU358 CA 1.515 12.727 10.168 2703 LEU358 C 1.271 11.264 9.826 2704 LEU358 O 2.234 10.487 9.791 2705 LEU358 CB 0.908 13.034 11.53 2706 LEU358 CG 1.612 12.261 12.639 2707 LEU358 CD1 3.089 12.639 12.712 2708 LEU358 CD2 0.931 12.493 13.982 2709 ALA359 N 0.102 10.986 9.272 2710 ALA359 CA −0.235 9.616 8.887 2711 ALA359 C 0.571 9.151 7.679 2712 ALA359 O 1.204 8.089 7.756 2713 ALA359 CB −1.723 9.558 8.566 2714 ARG360 N 0.794 10.045 6.728 2715 ARG360 CA 1.585 9.681 5.546 2716 ARG360 C 3.078 9.603 5.853 2717 ARG360 O 3.747 8.677 5.374 2718 ARG360 CB 1.354 10.721 4.459 2719 ARG360 CG −0.081 10.681 3.954 2720 ARG360 CD −0.325 11.752 2.898 2721 ARG360 NE −0.144 13.097 3.463 2722 ARG360 CZ 0.509 14.076 2.833 2723 ARG360 NH1 0.602 15.283 3.393 2724 ARG360 NH2 1.045 13.853 1.631 2725 LEU361 N 3.518 10.379 6.83 2726 LEU361 CA 4.921 10.356 7.239 2727 LEU361 C 5.238 9.078 8.005 2728 LEU361 O 6.174 8.36 7.628 2729 LEU361 CB 5.159 11.566 8.133 2730 LEU361 CG 6.612 11.69 8.572 2731 LEU361 CD1 7.537 11.811 7.365 2732 LEU361 CD2 6.778 12.888 9.498 2733 GLU362 N 4.306 8.658 8.846 2734 GLU362 CA 4.499 7.431 9.622 2735 GLU362 C 4.413 6.2 8.73 2736 GLU362 O 5.31 5.352 8.793 2737 GLU362 CB 3.418 7.347 10.694 2738 GLU362 CG 3.519 8.493 11.693 2739 GLU362 CD 2.341 8.447 12.662 2740 GLU362 OE1 1.268 8.901 12.284 2741 GLU362 OE2 2.517 7.891 13.736 2742 LEU363 N 3.541 6.253 7.736 2743 LEU363 CA 3.389 5.125 6.814 2744 LEU363 C 4.61 4.946 5.927 2745 LEU363 O 5.174 3.844 5.889 2746 LEU363 CB 2.184 5.364 5.914 2747 LEU363 CG 0.977 4.517 6.298 2748 LEU363 CD1 0.35 4.995 7.601 2749 LEU363 CD2 −0.056 4.54 5.179 2750 GLN364 N 5.151 6.047 5.432 2751 GLN364 CA 6.296 5.955 4.529 2752 GLN364 C 7.559 5.544 5.276 2753 GLN364 O 8.219 4.588 4.843 2754 GLN364 CB 6.505 7.308 3.86 2755 GLN364 CG 7.624 7.232 2.83 2756 GLN364 CD 7.846 8.592 2.181 2757 GLN364 OE1 7.741 9.637 2.835 2758 GLN364 NE2 8.108 8.565 0.886 2759 ILE365 N 7.707 6.019 6.503 2760 ILE365 CA 8.879 5.644 7.298 2761 ILE365 C 8.835 4.171 7.691 2762 ILE365 O 9.772 3.438 7.339 2763 ILE365 CB 8.94 6.511 8.554 2764 ILE365 CG1 9.202 7.973 8.208 2765 ILE365 CG2 10.012 5.996 9.506 2766 ILE365 CD1 10.539 8.151 7.497 2767 VAL366 N 7.667 3.695 8.099 2768 VAL366 CA 7.555 2.301 8.53 2769 VAL366 C 7.703 1.335 7.361 2770 VAL366 O 8.611 0.497 7.417 2771 VAL366 CB 6.21 2.076 9.217 2772 VAL366 CG1 6.011 0.606 9.565 2773 VAL366 CG2 6.085 2.923 10.477 2774 PHE367 N 7.094 1.641 6.225 2775 PHE367 CA 7.145 0.701 5.097 2776 PHE367 C 8.524 0.663 4.453 2777 PHE367 O 9.066 −0.434 4.254 2778 PHE367 CB 6.125 1.099 4.034 2779 PHE367 CG 4.662 1.012 4.458 2780 PHE367 CD1 3.726 1.83 3.841 2781 PHE367 CD2 4.258 0.112 5.435 2782 PHE367 CE1 2.391 1.767 4.216 2783 PHE367 CE2 2.924 0.052 5.813 2784 PHE367 CZ 1.99 0.88 5.206 2785 ASP368 N 9.187 1.808 4.423 2786 ASP368 CA 10.522 1.869 3.83 2787 ASP368 C 11.52 1.102 4.686 2788 ASP368 O 12.115 0.138 4.185 2789 ASP368 CB 10.964 3.326 3.707 2790 ASP368 CG 10.101 4.09 2.701 2791 ASP368 OD1 10.208 5.31 2.678 2792 ASP368 OD2 9.508 3.443 1.847 2793 THR369 N 11.438 1.285 5.995 2794 THR369 CA 12.381 0.606 6.892 2795 THR369 C 12.07 −0.88 7.076 2796 THR369 O 13.004 −1.658 7.287 2797 THR369 CB 12.376 1.294 8.252 2798 THR369 OG1 11.058 1.242 8.778 2799 THR369 CG2 12.797 2.756 8.149 2800 LEU370 N 10.848 −1.302 6.793 2801 LEU370 CA 10.523 −2.729 6.862 2802 LEU370 C 11.145 −3.486 5.703 2803 LEU370 O 12.038 −4.319 5.916 2804 LEU370 CB 9.012 −2.919 6.78 2805 LEU370 CG 8.302 −2.463 8.044 2806 LEU370 CD1 6.792 −2.589 7.882 2807 LEU370 CD2 8.79 −3.256 9.248 2808 PHE371 N 10.872 −3.006 4.502 2809 PHE371 CA 11.266 −3.75 3.303 2810 PHE371 C 12.728 −3.529 2.918 2811 PHE371 O 13.332 −4.398 2.278 2812 PHE371 CB 10.333 −3.34 2.169 2813 PHE371 CG 8.861 −3.634 2.463 2814 PHE371 CD1 7.935 −2.599 2.503 2815 PHE371 CD2 8.447 −4.94 2.692 2816 PHE371 CE1 6.601 −2.867 2.784 2817 PHE371 CE2 7.112 −5.209 2.971 2818 PHE371 CZ 6.19 −4.172 3.019 2819 ARG372 N 13.335 −2.472 3.436 2820 ARG372 CA 14.773 −2.281 3.231 2821 ARG372 C 15.606 −2.901 4.353 2822 ARG372 O 16.834 −2.982 4.229 2823 ARG372 CB 15.084 −0.795 3.116 2824 ARG372 CG 14.397 −0.176 1.904 2825 ARG372 CD 14.777 1.291 1.76 2826 ARG372 NE 14.497 2.015 3.008 2827 ARG372 CZ 14.919 3.257 3.251 2828 ARG372 NH1 14.646 3.835 4.423 2829 ARG372 NH2 15.631 3.911 2.331 2830 ARG373 N 14.958 −3.347 5.418 2831 ARG373 CA 15.671 −4.087 6.457 2832 ARG373 C 15.659 −5.559 6.09 2833 ARG373 O 16.694 −6.238 6.097 2834 ARG373 CB 14.937 −3.926 7.783 2835 ARG373 CG 15.657 −4.637 8.922 2836 ARG373 CD 16.912 −3.879 9.335 2837 ARG373 NE 16.548 −2.554 9.862 2838 ARG373 CZ 16.405 −2.304 11.165 2839 ARG373 NH1 16.663 −3.261 12.059 2840 ARG373 NH2 16.046 −1.087 11.576 2841 VAL374 N 14.474 −6.032 5.749 2842 VAL374 CA 14.314 −7.425 5.338 2843 VAL374 C 13.644 −7.499 3.971 2844 VAL374 O 12.43 −7.307 3.841 2845 VAL374 CB 13.473 −8.164 6.376 2846 VAL374 CG1 13.297 −9.625 5.984 2847 VAL374 CG2 14.086 −8.075 7.77 2848 PRO375 N 14.432 −7.873 2.976 2849 PRO375 CA 13.929 −8.036 1.606 2850 PRO375 C 13.088 −9.304 1.369 2851 PRO375 O 12.539 −9.472 0.275 2852 PRO375 CB 15.165 −8.072 0.76 2853 PRO375 CG 16.384 −8.221 1.658 2854 PRO375 CD 15.865 −8.159 3.084 2855 GLY376 N 12.945 −10.158 2.371 2856 GLY376 CA 12.162 −11.386 2.21 2857 GLY376 C 11.071 −11.52 3.271 2858 GLY376 O 11.012 −12.523 3.992 2859 ILE377 N 10.225 −10.508 3.367 2860 ILE377 CA 9.092 −10.568 4.299 2861 ILE377 C 7.921 −11.295 3.645 2862 ILE377 O 7.217 −10.731 2.801 2863 ILE377 CB 8.663 −9.148 4.656 2864 ILE377 CG1 9.836 −8.352 5.203 2865 ILE377 CG2 7.529 −9.169 5.674 2866 ILE377 CD1 9.433 −6.919 5.526 2867 ARG378 N 7.743 −12.55 4.009 2868 ARG378 CA 6.648 −13.342 3.451 2869 ARG378 C 5.468 −13.417 4.409 2870 ARG378 O 5.629 −13.304 5.627 2871 ARG378 CB 7.186 −14.734 3.163 2872 ARG378 CG 8.265 −14.671 2.089 2873 ARG378 CD 8.975 −16.01 1.929 2874 ARG378 NE 9.756 −16.33 3.134 2875 ARG378 CZ 9.57 −17.431 3.864 2876 ARG378 NH1 8.587 −18.28 3.556 2877 ARG378 NH2 10.338 −17.659 4.931 2878 ILE379 N 4.277 −13.531 3.854 2879 ILE379 CA 3.096 −13.713 4.703 2880 ILE379 C 3.14 −15.13 5.272 2881 ILE379 O 3.519 −16.07 4.563 2882 ILE379 CB 1.841 −13.536 3.855 2883 ILE379 CG1 2.108 −12.589 2.692 2884 ILE379 CG2 0.702 −12.984 4.709 2885 ILE379 CD1 0.882 −12.447 1.798 2886 ALA380 N 2.872 −15.267 6.56 2887 ALA380 CA 2.895 −16.598 7.174 2888 ALA380 C 1.533 −17.277 7.07 2889 ALA380 O 1.435 −18.51 7.097 2890 ALA380 CB 3.306 −16.471 8.635 2891 VAL381 N 0.498 −16.47 6.917 2892 VAL381 CA −0.839 −17.005 6.651 2893 VAL381 C −1.231 −16.745 5.2 2894 VAL381 O −0.782 −15.768 4.59 2895 VAL381 CB −1.847 −16.359 7.599 2896 VAL381 CG1 −1.705 −16.897 9.018 2897 VAL381 CG2 −1.747 −14.839 7.57 2898 PRO382 N −1.999 −17.662 4.635 2899 PRO382 CA −2.615 −17.424 3.329 2900 PRO382 C −3.477 −16.166 3.352 2901 PRO382 O −4.045 −15.802 4.391 2902 PRO382 CB −3.422 −18.651 3.039 2903 PRO382 CG −3.29 −19.627 4.198 2904 PRO382 CD −2.414 −18.938 5.231 2905 VAL383 N −3.721 −15.621 2.172 2906 VAL383 CA −4.415 −14.327 2.051 2907 VAL383 C −5.892 −14.388 2.452 2908 VAL383 O −6.376 −13.473 3.126 2909 VAL383 CB −4.302 −13.886 0.593 2910 VAL383 CG1 −5.05 −12.578 0.343 2911 VAL383 CG2 −2.838 −13.751 0.177 2912 ASP384 N −6.478 −15.572 2.355 2913 ASP384 CA −7.876 −15.767 2.759 2914 ASP384 C −8.031 −15.962 4.271 2915 ASP384 O −9.156 −16.094 4.761 2916 ASP384 CB −8.42 −17.003 2.048 2917 ASP384 CG −8.293 −16.849 0.534 2918 ASP384 OD1 −9.1 −16.128 −0.032 2919 ASP384 OD2 −7.312 −17.346 −0.002 2920 GLU385 N −6.926 −15.995 5 2921 GLU385 CA −6.994 −16.177 6.448 2922 GLU385 C −6.674 −14.884 7.194 2923 GLU385 O −6.638 −14.896 8.429 2924 GLU385 CB −6.01 −17.259 6.874 2925 GLU385 CG −6.219 −18.561 6.111 2926 GLU385 CD −7.651 −19.079 6.248 2927 GLU385 OE1 −8.017 −19.462 7.349 2928 GLU385 OE2 −8.266 −19.256 5.205 2929 LEU386 N −6.406 −13.81 6.463 2930 LEU386 CA −6.082 −12.519 7.093 2931 LEU386 C −7.266 −11.953 7.874 2932 LEU386 O −8.342 −11.71 7.315 2933 LEU386 CB −5.676 −11.542 5.996 2934 LEU386 CG −4.348 −11.943 5.365 2935 LEU386 CD1 −4.081 −11.153 4.091 2936 LEU386 CD2 −3.204 −11.773 6.357 2937 PRO387 N −7.063 −11.798 9.173 2938 PRO387 CA −8.132 −11.39 10.091 2939 PRO387 C −8.419 −9.89 10.047 2940 PRO387 O −7.84 −9.095 10.805 2941 PRO387 CB −7.647 −11.801 11.445 2942 PRO387 CG −6.191 −12.224 11.339 2943 PRO387 CD −5.817 −12.105 9.873 2944 PHE388 N −9.314 −9.528 9.143 2945 PHE388 CA −9.775 −8.145 9.012 2946 PHE388 C −10.688 −7.79 10.176 2947 PHE388 O −11.522 −8.597 10.603 2948 PHE388 CB −10.558 −7.999 7.709 2949 PHE388 CG −9.785 −8.343 6.437 2950 PHE388 CD1 −8.78 −7.498 5.987 2951 PHE388 CD2 −10.097 −9.492 5.721 2952 PHE388 CE1 −8.076 −7.809 4.831 2953 PHE388 CE2 −9.393 −9.804 4.565 2954 PHE388 CZ −8.381 −8.963 4.121 2955 LYS389 N −10.5 −6.599 10.707 2956 LYS389 CA −11.364 −6.141 11.792 2957 LYS389 C −12.626 −5.52 11.203 2958 LYS389 O −12.542 −4.581 10.4 2959 LYS389 CB −10.611 −5.115 12.633 2960 LYS389 CG −11.439 −4.708 13.847 2961 LYS389 CD −10.677 −3.763 14.767 2962 LYS389 CE −11.487 −3.466 16.023 2963 LYS389 NZ −10.719 −2.637 16.96 2964 HIS390 N −13.775 −6.068 11.571 2965 HIS390 CA −15.055 −5.523 11.102 2966 HIS390 C −15.385 −4.226 11.836 2967 HIS390 O −15.845 −4.213 12.983 2968 HIS390 CB −16.162 −6.548 11.316 2969 HIS390 CG −17.525 −6.094 10.826 2970 HIS390 ND1 −17.895 −5.893 9.545 2971 HIS390 CD2 −18.62 −5.81 11.607 2972 HIS390 CE1 −19.181 −5.487 9.511 2973 HIS390 NE2 −19.629 −5.437 10.786 2974 ASP391 N −15.053 −3.138 11.167 2975 ASP391 CA −15.269 −1.789 11.683 2976 ASP391 C −15.392 −0.871 10.48 2977 ASP391 O −14.395 −0.539 9.835 2978 ASP391 CB −14.068 −1.414 12.553 2979 ASP391 CG −14.172 −0.02 13.18 2980 ASP391 OD1 −14.984 0.771 12.707 2981 ASP391 OD2 −13.241 0.324 13.884 2982 SER392 N −16.582 −0.335 10.283 2983 SER392 CA −16.835 0.448 9.075 2984 SER392 C −16.261 1.863 9.129 2985 SER392 O −15.99 2.431 8.07 2986 SER392 CB −18.342 0.537 8.868 2987 SER392 OG −18.876 1.383 9.878 2988 THR393 N −15.944 2.392 10.297 2989 THR393 CA −15.451 3.768 10.313 2990 THR393 C −13.931 3.822 10.468 2991 THR393 O −13.294 4.722 9.909 2992 THR393 CB −16.183 4.53 11.411 2993 THR393 OG1 −17.557 4.561 11.04 2994 THR393 CG2 −15.703 5.972 11.528 2995 ILE394 N −13.365 2.816 11.118 2996 ILE394 CA −11.899 2.665 11.203 2997 ILE394 C −11.525 1.204 10.92 2998 ILE394 O −11.205 0.419 11.824 2999 ILE394 CB −11.37 3.095 12.577 3000 ILE394 CG1 −11.744 4.533 12.921 3001 ILE394 CG2 −9.847 2.978 12.624 3002 ILE394 CD1 −10.977 5.529 12.055 3003 TYR395 N −11.59 0.854 9.649 3004 TYR395 CA −11.29 −0.503 9.179 3005 TYR395 C −9.792 −0.786 9.302 3006 TYR395 O −8.997 0.149 9.447 3007 TYR395 CB −11.747 −0.572 7.721 3008 TYR395 CG −11.784 −1.967 7.101 3009 TYR395 CD1 −10.958 −2.272 6.026 3010 TYR395 CD2 −12.648 −2.927 7.612 3011 TYR395 CE1 −10.991 −3.543 5.465 3012 TYR395 CE2 −12.682 −4.199 7.052 3013 TYR395 CZ −11.852 −4.502 5.982 3014 TYR395 OH −11.882 −5.763 5.427 3015 GLY396 N −9.433 −2.053 9.421 3016 GLY396 CA −8.007 −2.401 9.468 3017 GLY396 C −7.732 −3.871 9.759 3018 GLY396 O −8.601 −4.74 9.609 3019 LEU397 N −6.493 −4.132 10.134 3020 LEU397 CA −6.031 −5.497 10.406 3021 LEU397 C −5.334 −5.577 11.751 3022 LEU397 O −4.297 −4.938 11.961 3023 LEU397 CB −5.051 −5.894 9.311 3024 LEU397 CG −5.773 −6.502 8.12 3025 LEU397 CD1 −5.037 −6.225 6.822 3026 LEU397 CD2 −5.979 −7.996 8.325 3027 HIS398 N −5.87 −6.402 12.634 3028 HIS398 CA −5.274 −6.514 13.967 3029 HIS398 C −4.348 −7.718 14.107 3030 HIS398 O −3.651 −7.848 15.12 3031 HIS398 CB −6.363 −6.528 15.033 3032 HIS398 CG −6.737 −5.14 15.525 3033 HIS398 ND1 −7.052 −4.804 16.79 3034 HIS398 CD2 −6.795 −3.984 14.781 3035 HIS398 CE1 −7.311 −3.482 16.851 3036 HIS398 NE2 −7.152 −2.975 15.607 3037 ALA399 N −4.306 −8.567 13.094 3038 ALA399 CA −3.343 −9.671 13.12 3039 ALA399 C −2.7 −9.903 11.756 3040 ALA399 O −3.373 −10.014 10.724 3041 ALA399 CB −4.014 −10.936 13.633 3042 LEU400 N −1.383 −10.004 11.794 3043 LEU400 CA −0.567 −10.204 10.589 3044 LEU400 C 0.772 −10.856 10.935 3045 LEU400 O 1.712 −10.165 11.35 3046 LEU400 CB −0.307 −8.845 9.946 3047 LEU400 CG 0.615 −8.952 8.736 3048 LEU400 CD1 0.005 −9.826 7.644 3049 LEU400 CD2 0.973 −7.572 8.197 3050 PRO401 N 0.815 −12.178 10.881 3051 PRO401 CA 2.084 −12.9 10.974 3052 PRO401 C 2.88 −12.822 9.671 3053 PRO401 O 2.413 −13.242 8.602 3054 PRO401 CB 1.686 −14.312 11.269 3055 PRO401 CG 0.197 −14.466 10.993 3056 PRO401 CD −0.308 −13.08 10.625 3057 VAL402 N 4.074 −12.267 9.78 3058 VAL402 CA 5.008 −12.183 8.653 3059 VAL402 C 6.358 −12.814 8.998 3060 VAL402 O 7.008 −12.485 9.998 3061 VAL402 CB 5.194 −10.723 8.25 3062 VAL402 CG1 3.968 −10.185 7.523 3063 VAL402 CG2 5.553 −9.84 9.44 3064 THR403 N 6.772 −13.729 8.146 3065 THR403 CA 8.039 −14.428 8.342 3066 THR403 C 9.135 −13.709 7.571 3067 THR403 O 9.102 −13.66 6.335 3068 THR403 CB 7.888 −15.853 7.827 3069 THR403 OG1 6.715 −16.403 8.406 3070 THR403 CG2 9.077 −16.723 8.22 3071 TRP404 N 10.089 −13.156 8.298 3072 TRP404 CA 11.177 −12.406 7.66 3073 TRP404 C 12.136 −13.344 6.931 3074 TRP404 O 12.984 −12.835 6.21 3075 TRP404 CB 11.969 −11.654 8.719 3076 TRP404 CG 11.163 −10.949 9.79 3077 TRP404 CD1 10.886 −11.444 11.043 3078 TRP404 CD2 10.559 −9.637 9.729 3079 TRP404 NE1 10.155 −10.524 11.721 3080 TRP404 CE2 9.943 −9.428 10.972 3081 TRP404 CE3 10.506 −8.656 8.749 3082 TRP404 CZ2 9.278 −8.237 11.225 3083 TRP404 CZ3 9.838 −7.468 9.009 3084 TRP404 CH2 9.226 −7.257 10.239 3085 TRP404 OXT 12.117 −14.53 7.239 3086 HEM1 FE −8.08 12.05 10.226 3087 HEM1 NA −9.653 12.085 9.078 3088 HEM1 C1A −10.7 13.004 9.077 3089 HEM1 C2A −11.687 12.681 8.118 3090 HEM1 C3A −11.292 11.525 7.568 3091 HEM1 C4A −10.019 11.174 8.129 3092 HEM1 CHB −9.224 10.115 7.699 3093 HEM1 C1B −7.931 9.83 8.181 3094 HEM1 NB −7.308 10.582 9.182 3095 HEM1 C4B −6.086 9.964 9.364 3096 HEM1 C3B −5.946 8.85 8.506 3097 HEM1 C2B −7.068 8.771 7.746 3098 HEM1 CMB −7.416 7.755 6.682 3099 HEM1 CAB −4.833 8.031 8.591 3100 HEM1 CBB −4.44 7.051 7.74 3101 HEM1 CHC −5.212 10.298 10.374 3102 HEM1 C1C −5.439 11.223 11.336 3103 HEM1 NC −6.519 12.039 11.384 3104 HEM1 C4C −6.227 12.887 12.426 3105 HEM1 C3C −4.926 12.636 13.002 3106 HEM1 C2C −4.491 11.556 12.313 3107 HEM1 CMC −3.265 10.712 12.532 3108 HEM1 CAC −4.462 13.435 14.055 3109 HEM1 CBC −3.452 13.231 14.936 3110 HEM1 CHD −7.061 13.855 12.91 3111 HEM1 C1D −8.237 14.203 12.292 3112 HEM1 ND −8.777 13.572 11.18 3113 HEM1 C4D −9.915 14.313 10.916 3114 HEM1 C3D −10.045 15.413 11.808 3115 HEM1 C2D −9.006 15.334 12.673 3116 HEM1 CMD −8.71 16.241 13.844 3117 HEM1 CAD −11.178 16.421 11.802 3118 HEM1 CBD −10.91 17.624 10.918 3119 HEM1 CGD −12.079 18.574 10.862 3120 HEM1 O1D −13.198 18.167 11.204 3121 HEM1 O2D −11.889 19.736 10.477 3122 HEM1 CHA −10.849 14.026 9.961 3123 HEM1 CMA −12.005 10.703 6.498 3124 HEM1 CAA −12.907 13.51 7.748 3125 HEM1 CBA −14.087 13.112 8.645 3126 HEM1 CGA −15.442 13.596 8.14 3127 HEM1 O1A −15.522 14.131 7.009 3128 HEM1 O2A −16.439 13.4 8.866 

1. An isolated nucleic acid sequence encoding epothilone B hydroxylase of SEQ ID NO: 2 or a mutant thereof, wherein said mutant comprises at least one amino acid substitution at an amino acid position seleted from the group consisting of GLU31, ARG67, ARG88, ILE92, ALA93, VAL106, ILE130, ALA140, MET176, PHE190, GLU231, SER294, PHE237, and ILE365 of SEQ ID NO: 2, and wherein said mutant has epothilone B hydroxylase activity.
 2. The isolated nucleic acid sequence of claim 1 comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 30, 32, 34, 60, 62, 64, 66, 68, 72 and
 74. 3. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at GLU31 of SEQ ID NO:2.
 4. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid sequence substitution at ARG67 of SEQ ID NO:2.
 5. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at ARG88 of SEQ ID NO:2.
 6. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at ILE92 of SEQ ID NO:2.
 7. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at ALA93 of SEQ ID NO:2.
 8. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at VAL106 of SEQ ID NO:2.
 9. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at ILE130 of SEQ ID NO:2.
 10. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at ALA140 of SEQ ID NO:2.
 11. The isolated nucleic avid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at MET176 of SEQ ID NO:2.
 12. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at PHE190 of SEQ ID NO:2.
 13. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at GLU231 of SEQ ID NO:2.
 14. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at SER294 of SEQ ID NO:2.
 15. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at PHE237 of SBQ ID NO:2.
 16. The isolated nucleic acid sequence of claim 1 wherein said at least one mutation comprises an amino acid substitution at ILE365 of SEQ ID NO:2.
 17. A vector comprising the isolated nucleic acid sequence of claim
 1. 18. An isolated host cell comprising the vector of claim
 17. 19. A method for producing a recombinant microorganism which hydroxylate epothilone B, said method comprising transfecting a microorganism with the vector of claim
 17. 20. A recombinant microorganism produced by the method of claim
 19. 21. The recombinant microorganism of claim 20 wherein said microorganism expresses a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 30, 32, 34, 60, 62, 64, 66, 68, 72 and
 74. 